Anti-TIGIT antibodies, anti-PVRIG antibodies and combinations thereof

ABSTRACT

Anti-PVRIG and anti-TIGIT antibodies are provided.

I. RELATED APPLICATIONS

This application claims priority to U.S. Application Ser. No.62/376,334, filed on Aug. 17, 2016, U.S. Application Ser. No. 62/513,771filed on Jun. 1, 2017, U.S. Application Ser. No. 62/376,335, filed onAug. 17, 2016, U.S. Application Ser. No. 62/417,217, filed on Nov. 3,2016, U.S. Application Ser. No. 62/513,775, filed on Jun. 1, 2017, U.S.Application Ser. No. 62/477,974, filed on Mar. 28, 2017, U.S.Application Ser. No. 62/513,916, filed on Jun. 1, 2017, and U.S.Application Ser. No. 62/538,561, filed on Jul. 28, 2017, all of whichare incorporated by reference herein in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 17, 2017 andamended on Mar. 1, 2018, is named 114386-5008-WO_SL.txt and is 593,346bytes in size.

II. BACKGROUND OF THE INVENTION

Naïve T cells must receive two independent signals fromantigen-presenting cells (APC) in order to become productivelyactivated. The first, Signal 1, is antigen-specific and occurs when Tcell antigen receptors encounter the appropriate antigen-MHC complex onthe APC. The fate of the immune response is determined by a second,antigen-independent signal (Signal 2) which is delivered through a Tcell costimulatory molecule that engages its APC-expressed ligand. Thissecond signal could be either stimulatory (positive costimulation) orinhibitory (negative costimulation or coinhibition). In the absence of acostimulatory signal, or in the presence of a coinhibitory signal,T-cell activation is impaired or aborted, which may lead to a state ofantigen-specific unresponsiveness (known as T-cell anergy), or mayresult in T-cell apoptotic death.

Costimulatory molecule pairs usually consist of ligands expressed onAPCs and their cognate receptors expressed on T cells. The prototypeligand/receptor pairs of costimulatory molecules are B7/CD28 andCD40/CD40L. The B7 family consists of structurally related, cell-surfaceprotein ligands, which may provide stimulatory or inhibitory input to animmune response. Members of the B7 family are structurally related, withthe extracellular domain containing at least one variable or constantimmunoglobulin domain.

Both positive and negative costimulatory signals play critical roles inthe regulation of cell-mediated immune responses, and molecules thatmediate these signals have proven to be effective targets forimmunomodulation. Based on this knowledge, several therapeuticapproaches that involve targeting of costimulatory molecules have beendeveloped, and were shown to be useful for prevention and treatment ofcancer by turning on, or preventing the turning off, of immune responsesin cancer patients and for prevention and treatment of autoimmunediseases and inflammatory diseases, as well as rejection of allogenictransplantation, each by turning off uncontrolled immune responses, orby induction of “off signal” by negative costimulation (or coinhibition)in subjects with these pathological conditions.

Manipulation of the signals delivered by B7 ligands has shown potentialin the treatment of autoimmunity, inflammatory diseases, and transplantrejection. Therapeutic strategies include blocking of costimulationusing monoclonal antibodies to the ligand or to the receptor of acostimulatory pair, or using soluble fusion proteins composed of thecostimulatory receptor that may bind and block its appropriate ligand.Another approach is induction of co-inhibition using soluble fusionprotein of an inhibitory ligand. These approaches rely, at leastpartially, on the eventual deletion of auto- or allo-reactive T cells(which are responsible for the pathogenic processes in autoimmunediseases or transplantation, respectively), presumably because in theabsence of costimulation (which induces cell survival genes) T cellsbecome highly susceptible to induction of apoptosis. Thus, novel agentsthat are capable of modulating costimulatory signals, withoutcompromising the immune system's ability to defend against pathogens,are highly advantageous for treatment and prevention of suchpathological conditions.

Costimulatory pathways play an important role in tumor development.Interestingly, tumors have been shown to evade immune destruction byimpeding T cell activation through inhibition of co-stimulatory factorsin the B7-CD28 and TNF families, as well as by attracting regulatory Tcells, which inhibit anti-tumor T cell responses (see Wang (2006),“Immune Suppression by Tumor Specific CD4⁺ Regulatory T cells inCancer”, Semin. Cancer. Biol. 16:73-79; Greenwald, et al. (2005), “TheB7 Family Revisited”, Ann. Rev. Immunol. 23:515-48; Watts (2005),“TNF/TNFR Family Members in Co-stimulation of T Cell Responses”, Ann.Rev. Immunol. 23:23-68; Sadum, et al., (2007) “Immune Signatures ofMurine and Human Cancers Reveal Unique Mechanisms of Tumor Escape andNew Targets for Cancer Immunotherapy”, Clin. Canc. Res. 13(13):4016-4025). Such tumor expressed co-stimulatory molecules have becomeattractive cancer biomarkers and may serve as tumor-associated antigens(TAAs). Furthermore, costimulatory pathways have been identified asimmunologic checkpoints that attenuate T cell dependent immuneresponses, both at the level of initiation and effector function withintumor metastases. As engineered cancer vaccines continue to improve, itis becoming clear that such immunologic checkpoints are a major barrierto the vaccines' ability to induce therapeutic anti-tumor responses. Inthat regard, costimulatory molecules can serve as adjuvants for active(vaccination) and passive (antibody-mediated) cancer immunotherapy,providing strategies to thwart immune tolerance and stimulate the immunesystem.

Over the past decade, agonists and/or antagonists to variouscostimulatory proteins have been developed for treating autoimmunediseases, graft rejection, allergy and cancer. For example, CTLA4-Ig(Abatacept, Orencia®) is approved for treatment of RA, mutated CTLA4-Ig(Belatacept, Nulojix®) for prevention of acute kidney transplantrejection and by the anti-CTLA4 antibody (Ipilimumab, Yervoy®), recentlyapproved for the treatment of melanoma. Other costimulation regulatorshave been approved, such as the anti-PD-1 antibodies of Merck(Keytruda®) and BMS (Opdivo®), have been approved for cancer treatmentsand are in testing for viral infections as well.

However, while monotherapy with anti-checkpoint inhibitor antibodieshave shown promise, a number of studies (Ahmadzadeh et al., Blood114:1537 (2009), Matsuzaki et al., PNAS 107(17):7875-7880 (2010),Fourcade et al., Cancer Res. 72(4):887-896 (2012) and Gros et al., J.Clinical Invest. 124(5):2246 (2014)) examining tumor-infiltratinglymphocytes (TILs) have shown that TILs commonly express multiplecheckpoint receptors. Moreover, it is likely that TILs that expressmultiple checkpoints are in fact the most tumor-reactive. In contrast,non-tumor reactive T cells in the periphery are more likely to express asingle checkpoint. Checkpoint blockade with monospecific full-lengthantibodies is likely nondiscriminatory with regards to de-repression oftumor-reactive TILs versus autoantigen-reactive single expressing Tcells that are assumed to contribute to autoimmune toxicities.

One target of interest is PVRIG. PVRIG, also called Poliovirus ReceptorRelated Immunoglobulin Domain Containing Protein, Q6DKI7 or C7orf15, isa transmembrane domain protein of 326 amino acids in length, with asignal peptide (spanning from amino acid 1 to 40), an extracellulardomain (spanning from amino acid 41 to 171), a transmembrane domain(spanning from amino acid 172 to 190) and a cytoplasmic domain (spanningfrom amino acid 191 to 326). PVRIG binds to Poliovirus receptor-related2 protein (PVLR2, also known as nectin-2, CD112 or herpesvirus entrymediator B, (HVEB) a human plasma membrane glycoprotein), the bindingpartner of PVRIG.

Another target of interest is TIGIT. TIGIT is a coinhibitory receptorthat is highly expressed on effector & regulatory (Treg) CD4+ T cells,effector CD8+ T cells, and NK cells. TIGIT has been shown to attenuateimmune response by (1) direct signaling, (2) inducing ligand signaling,and (3) competition with and disruption of signaling by thecostimulatory receptor CD226 (also known as DNAM-1). TIGIT signaling hasbeen the most well-studied in NK cells, where it has been demonstratedthat engagement with its cognate ligand, poliovirus receptor (PVR, alsoknown as CD155) directly suppresses NK cell cytotoxicity through itscytoplasmic ITIM domain. Knockout of the TIGIT gene or antibody blockadeof the TIGIT/PVR interaction has shown to enhance NK cell killing invitro, as well as to exacerbate autoimmune diseases in vivo. In additionto its direct effects on T- and NK cells, TIGIT can induce PVR-mediatedsignaling in dendritic or tumor cells, leading to the increase inproduction of anti-inflammatory cytokines such as IL10. In T-cells TIGITcan also inhibit lymphocyte responses by disrupting homodimerization ofthe costimulatory receptor CD226, and by competing with it for bindingto PVR.

TIGIT is highly expressed on lymphocytes, including Tumor InfiltratingLymphocytes (TILs) and Tregs, that infiltrate different types of tumors.PVR is also broadly expressed in tumors, suggesting that the TIGIT-PVRsignaling axis may be a dominant immune escape mechanism for cancer.Notably, TIGIT expression is tightly correlated with the expression ofanother important coinhibitory receptor, PD1. TIGIT and PD1 areco-expressed on the TILs of numerous human and murine tumors. UnlikeTIGIT and CTLA4, PD1 inhibition of T cell responses does not involvecompetition for ligand binding with a costimulatory receptor.

Accordingly, TIGIT is an attractive target for monoclonal antibodytherapy, and in addition in combination with additional antibodiesincluding anti-PVRIG antibodies.

III. BRIEF SUMMARY OF THE INVENTION

Accordingly, in one aspect, the invention provides compositionscomprising an antigen binding domain that binds to human TIGIT (SEQ IDNO:97) comprising a variable heavy domain comprising SEQ ID NO:160 and avariable light domain comprising SEQ ID NO:165. Additionally, theantigen binding domain comprises a variable heavy domain comprising SEQID NO:150 and a variable light domain comprising SEQ ID NO:155.Additionally, the antigen binding domain comprises a variable heavydomain comprising SEQ ID NO:560 and a variable light domain comprisingSEQ ID NO:565.

In a further aspect, the invention provides composition comprisingantibodies comprising a heavy chain comprising VH-CH1-hinge-CH2-CH3,wherein said VH comprises SEQ ID NO:160 and a light chain comprisingVL-VC, wherein said VL comprising SEQ ID NO:165 and VC is either kappaor lambda. Additionally, the antibody can comprise a heavy chaincomprising VH-CH1-hinge-CH2-CH3, wherein the VH comprises SEQ ID NO:150;and a light chain comprising VL-VC, wherein said VL comprising SEQ IDNO:159 and VC is either kappa or lambda. Additionally, the antibody cancomprise a heavy chain comprising VH-CH1-hinge-CH2-CH3, wherein the VHcomprises SEQ ID NO:560; and a light chain comprising VL-VC, whereinsaid VL comprising SEQ ID NO:565 and VC is either kappa or lambda.

In some aspects, the sequence of the CH1-hinge-CH2-CH3 is selected fromhuman IgG1, IgG2 and IgG4, and variants thereof. In some aspects, theheavy chain has SEQ ID NO:164 and the light chain has SEQ ID NO:169.

In an additional aspect, the compositions can further comprise a secondantibody that binds to a human checkpoint receptor protein, which can behuman PD-1 or human PVRIG. The second antibody can comprise an antigenbinding domain comprising a variable heavy domain comprising SEQ ID NO:5and a variable light domain comprising SEQ ID NO:10, or a heavy chainhaving SEQ ID NO:9 and a light chain having SEQ ID NO:14.

In a further aspect, the invention provides nucleic acid compositionscomprising a first nucleic acid encoding a variable heavy domaincomprising SEQ ID NO:160 and a second nucleic acid encoding a variablelight domain comprising SEQ ID NO:165. Alternatively, the nucleic acidcompositions comprise a first nucleic acid encoding a variable heavydomain comprising SEQ ID NO:150 and a second nucleic acid encoding avariable light domain comprising SEQ ID NO:155. Alternatively, thenucleic acid compositions comprise a first nucleic acid encoding avariable heavy domain comprising SEQ ID NO:560 and a second nucleic acidencoding a variable light domain comprising SEQ ID NO:565.

In a further aspect, the invention provides expression vectorcompositions comprising these nucleic acid compositions are provided aswell, such as a first expression vector comprising a first nucleic acidand a second expression vector comprising a second nucleic acid, oralternatively an expression vector that comprises both first and secondnucleci acids.

In an additional aspect, the invention provides host cells comprisingthe expression vector compositions, and methods of making the antibodiescomprising culturing the host cells under conditions wherein theantibodies are produced and recovering the antibody.

In a further aspect the invention provides anti-PVRIG antibodiescomprising a heavy chain having SEQ ID NO:9 and a light chain having SEQID NO:14. The invention further provides antibodies having a heavy chainhaving SEQ ID NO:19; and a light chain having SEQ ID NO:24.

In an additional aspect, an anti-PVRIG antibody (eitherCHA.7.518.1.H4(S241P) or CHA.7.538.1.2.H4(S241P) are co-administeredwith a second antibody that binds to a human checkpoint receptorprotein, such as an antibody that binds PD-1.

In a further aspect, an anti-PVRIG antibody (eitherCHA.7.518.1.H4(S241P) or CHA.7.538.1.2.H4(S241P)) are co-administeredwith a second antibody that binds to a human checkpoint receptorprotein, such as an antibody that binds human TIGIT, such as CPA.9.086or CPA.9.083 or CHA.9.547.13.

In a further aspect, the invention provides nucleic acid compositionscomprising a first nucleic acid encoding the heavy chain of eitherCHA.7.518.1.H4(S241P) or CHA.7.538.1.2.H4(S241P)) and a second nucleicacid encoding the light chain of either CHA.7.518.1.H4(S241P) orCHA.7.538.1.2.H4(S241P), respectively.

In a further aspect, the invention provides expression vectorcompositions comprising these nucleic acid compositions are provided aswell, such as a first expression vector comprising a first nucleic acidand a second expression vector comprising a second nucleic acid, oralternatively an expression vector that comprises both first and secondnucleci acids.

In an additional aspect, the invention provides host cells comprisingthe expression vector compositions, and methods of making the antibodiescomprising culturing the host cells under conditions wherein theantibodies are produced and recovering the antibody.

In a further aspect, the invention provides methods comprising: a)providing a cell population from a tumor sample from a patient; b)staining said population with labeled antibodies that bind: i) TIGITprotein; ii) PVR protein; iii) PD-1 protein; iv) PD-L1 protein; and v)an isotype control; c) running fluorescence activated cell sorting(FACS); d) for each of TIGIT, PVR, PD-1 and PD-L1, determining thepercentage of cells in said population that express the protein relativeto said isotype control antibody; wherein if the percentage of positivecells is ≥1% for all 4 receptors, e) administering antibodies to TIGITand PD-1 to said patient.

In an additional aspect, the invention provides methods comprising: a)providing a cell population from a tumor sample from a patient; b)staining said population with labeled antibodies that bind: i) PVRIGprotein; ii) PVRL2 protein; iii) PD-1 protein; iv) PD-L1 protein; and v)an isotype control; c) running fluorescence activated cell sorting(FACS); d) for each of PVRIG, PVRL2, PD-1 and PD-L1, determining thepercentage of cells in said population that express the protein relativeto said isotype control antibody; wherein if the percentage of positivecells is ≥1% for all 4 receptors, e) administering antibodies to PVRIGand PD-1 to said patient.

In a further aspect, the invention provides methods comprising a)providing a cell population from a tumor sample from a patient; b)staining said population with labeled antibodies that bind: i) PVRIGprotein; ii) PVRL2 protein; iii) TIGIT protein; iv) PVR protein; and v)an isotype control; c) running fluorescence activated cell sorting(FACS); d) for each of PVRIG, PVRL2, TIGIT and PVR, determining thepercentage of cells in said population that express the protein relativeto said isotype control antibody; wherein if the percentage of positivecells is ≥1% for all 4 receptors, e) administering antibodies to PVRIGand TIGIT to said patient.

IV. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the full-length sequence of human PVRIG (showing twodifferent methionine starting points). The signal peptide is underlined,the ECD is double underlined. PVRIG, also called Poliovirus ReceptorRelated Immunoglobulin Domain Containing Protein, Q6DKI7 or C7orf15,relates to amino acid and nucleic acid sequences shown in RefSeqaccession identifier NP_076975, shown in FIG. 1.

FIG. 2 depicts the sequence of the human Poliovirus receptor-related 2protein (PVLR2, also known as nectin-2, CD112 or herpesvirus entrymediator B, (HVEB)), the binding partner of PVRIG. PVLR2 is a humanplasma membrane glycoprotein.

FIGS. 3A and 3B depicts the variable heavy and light chains as well asthe vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2 and vlCDR3 sequences of eachof the enumerated CHA antibodies of the invention,CHA.7.518.1.H4(S241P), and CHA.7.538.1.2.H4(S241P).

FIGS. 4A and 4B PVRIG antibodies increase T cell proliferation in theMLR. The percentages of CFSE low cells are shown from MLR assays treatedwith the indicated PVRIG antibodies. Each graph represents oneindividual CD3 T cell donor. The experiments are described in Example 23of U.S. Ser. No. 15/048,967, incorporated by reference herein.

FIGS. 5A and 5B PVRIG hybridoma antibody binding characteristics to HEKhPVRIG engineered cell lines, HEK parental cells, and Jurkat cells. HEKOE denotes HEK hPVRIG cells, HEK par denotes HEK parental cells. ForJurkat data, gMFIr indicates the fold difference in geometric MFI ofPVRIG antibody staining relative to their controls. Concentrationindicates that at which the gMFIr was calculated. No binding indicatesantibody does not bind to the tested cell line. Highlighted antibodiesare the ‘top four’ antibodies of interest.

FIGS. 6A and 6B PVRIG hybridoma antibody binding characteristics toprimary human PBMC, cyno over-expressing cells, and cyno primary PBMC.Expi cyno OE denotes expi cells transiently transfected with cPVRIG,expi par denotes expi parental cells. gMFIr indicates the folddifference in geometric MFI of PVRIG antibody staining relative to theircontrols. Concentrations indicate that at which the gMFIr wascalculated. Not tested indicates antibodies that were not tested due toan absence of binding to human HEK hPVRIG, expi cPVRIG cells, or notmeeting binding requirements to PBMC subsets. Highlighted antibodies arethe ‘top four’ antibodies of interest. The experiments are described inExample 21 of U.S. Ser. No. 15/048,967, incorporated by referenceherein.

FIGS. 7A and 7B Summary of blocking capacity of PVRIG antibodies in theFACS-based competition assay. The IC₅₀ of inhibition is indicated. NoIC₅₀ indicates that these antibodies are non-blockers. Highlightedantibodies are the ‘top four’ antibodies of interest. The experimentsare described in Example 21 of U.S. Ser. No. 15/048,967, incorporated byreference herein.

FIGS. 8A and 8B TILs were co-cultured with melanoma cells 624 at 1:1 E:Tfor 18 hr in the presence of anti-PVRIG Ab (CPA.7.021; 10 ug/ml),anti-TIGIT (10A7 clone; 10 ug/ml) or in combination. Supernatant wascollected and tested in Th1 Th2 Th17 cytometric bead array assay todetect secreted cytokines. IFNγ (A) and TNF (B) levels were detected.Treatments were compared by Student's t-test (*P≤0.05, **P≤0.01) oftriplicate samples.

FIG. 9A-9F MART-1 or 209 TILs were co-cultured with melanoma cells 624at 1:1 E:T for 18 hr in the presence of anti-PVRIG Ab (CPA.7.021; 10ug/ml), anti-DNAM1 (DX11 clone, BD Biosciences Cat. No. 559787; 10ug/ml) or in combination. Supernatant was collected and tested in Th1Th2 Th17 cytometric bead array assay to detect secreted cytokines. IFNγ(A,D) and TNF (B,E) levels were detected. TILs were stained for surfaceexpression of CD137 (C,F).

FIGS. 10A and 10B TILs (F4) were co-cultured with melanoma cells 624 at1:3 E:T for 18 hr in the presence of anti-PVRIG Ab (CPA.7.021; 10ug/ml), anti-TIGIT (10A7 clone; 10 ug/ml), anti-PD1 (mAb 1B8, Merck; 10ug/ml) or in combination. Supernatant was collected and tested in Th1Th2 Th17 cytometric bead array assay to detect secreted cytokines. IFNγ(A) and TNF (B) levels were detected.

FIG. 11A-11E depict four humanized sequences for each of CHA.7.518,CHA.7.524, CHA.7.530, CHA.7.538_1 and CHA.7.538_2. All humanizedantibodies comprise the H4(S241P) substitution. Note that the lightchain for CHA.7.538_2 is the same as for CHA.7.538_1. The “H1” of eachis a “CDR swap” with no changes to the human framework. Subsequentsequences alter framework changes shown in larger bold font. CDRsequences are noted in bold. CDR definitions are AbM from websitewww.biOinf.org.uk/abs/. Human germline and joining sequences from IMGT®the international ImMunoGeneTics® information system www.imgt.org(founder and director: Marie-Paule Lefranc, Montpellier, France).Residue numbering shown as sequential (seq) or according to Chothia fromwebsite www.bioinf org.uk/abs/(AbM). “b” notes buried sidechain; “p”notes partially buried; “i” notes sidechain at interface between VH andVL domains. Sequence differences between human and murine germlinesnoted by asterisk (*). Potential additional mutations in frameworks arenoted below sequence. Potential changes in CDR sequences noted beloweach CDR sequence as noted on the figure (# deamidation substitutions:Q/S/A; these may prevent asparagine (N) deamidation. @ tryptophanoxidation substitutions: Y/F/H; these may prevent tryptophan oxidation;@ methionine oxidation substitutions: L/F/A).

FIG. 12A to 12C depicts a collation of the humanized sequences of threeCHA antibodies: CHA.7.518, CHA.7.538.1, and CHA.7.538.2.

FIG. 13 depicts schemes for combining the humanized VH and VL CHAantibodies. The “chimVH” and “chimVL” are the mouse variable heavy andlight sequences attached to a human IgG constant domain.

FIG. 14. PVRIG hybridoma antibody binding characteristics to primaryhuman PBMC, cyno over-expressing cells, and cyno primary PBMC. Expi cynoOE denotes expi cells transiently transfected with cPVRIG, expi pardenotes expi parental cells. gMFIr indicates the fold difference ingeometric MFI of PVRIG antibody staining relative to their controls.Concentrations indicate that at which the gMFIr was calculated. Nottested indicates antibodies that were not tested due to an absence ofbinding to human HEK hPVRIG, expi cPVRIG cells, or not meeting bindingrequirements to PBMC subsets. Highlighted antibodies are four antibodiesfor which humanization was done (See FIG. 24). The experiments aredescribed in Example 21 of U.S. Ser. No. 15/048,967, incorporated byreference herein.

FIG. 15. Summary of blocking capacity of PVRIG antibodies in theFACS-based competition assay. The IC50 of inhibition is indicated. NoIC50 indicates that these antibodies are non-blockers. Highlightedantibodies are four antibodies for which humanization was done (See FIG.24).

FIG. 16. Summary of the activity of select PVRIG antibodies in NK cellcytotoxicity assays against Reh and MOLM-13 cells. Fold change incytotoxicity relative to control was calculated by dividing the absolutelevel of killing (%) in the condition with PVRIG antibody, by theabsolute level of killing (%) with control antibody. Fold change iscalculated from the 5:1 effector to target ratio.

FIG. 17. Sequence alignment of PVRIG orthologs. Aligned sequences of thehuman, cynomolgus, marmoset, and rhesus PVRIG extra-cellular domain. Thedifferences between human and cynomolgus are highlighted in yellow.

FIG. 18A-18F. Binding of anti-human PVRIG antibodies to cyno, human,cyno/human hybrid PVRIG variants. Binding of antibodies to wild typecyno PVRIG (●), H61R cyno PVRIG (▪), P67S cyno PVRIG (▴), L95R/T97I cynoPVRIG (▾), and wild type human PVRIG (♦) are shown. The ELISA signalsare plotted as a function of antibody concentration.

FIG. 19. Correlation of epitope group and cyno cross-reactivity ofanti-human PVRIG antibodies.

FIGS. 20A and 20B (A) Specificity of CHA.7.518.1.H4(S241P) towards HEKcells engineered to overexpress PVRIG and HEK parental cells. Data showsabsolute geometric MFI (gMFI) measurements as a function of increasingantibody concentration. (B) Specificity of CHA.7.538.1.2.H4(S241P)towards HEK cells engineered to overexpress PVRIG and HEK parentalcells. Data shows absolute geometric MFI (gMFI) measurements as afunction of increasing antibody concentration.

FIGS. 21A and 21B illustrates the ability of CHA.7.518.1.H4(S241P) (A)and CHA.7.538.1.2.H4(S241P) (B) to bind Jurkat cells that endogenouslyexpress PVRIG confirmed by RNA expression. (A) Binding ofCHA.7.518.1.H4(S241P) to Jurkat cells. Data shows absolute geometric MFI(gMFI) measurements as a function of increasing antibody concentration.Isotype staining is shown as a negative control. (B) Binding ofCHA.7.538.1.2.H4(S241P) to Jurkat cells. Data shows absolute geometricMFI (gMFI) measurements as a function of increasing antibodyconcentration. Isotype staining is shown as a negative control. Bothantibodies are able to bind Jurkat cells with a comparable affinity toHEK hPVRIG cells.

FIG. 22 illustrates the ability of CHA.7.518.1.H4(S241P) andCHA.7.538.1.2.H4(S241P) to bind CD8 T cells that were expanded byexposure to CMV peptide (494-503, NLVPMVATV) and endogenously expressPVRIG confirmed by RNA expression. Binding of CHA.7.518.1.H4(S241P) andCHA.7.538.1.2.H4(S241P) to CMV peptide-expanded CD8 T cells. Data showsabsolute geometric MFI (gMFI) measurements as a function of increasingantibody concentration. Isotype staining is shown as a negative control.

FIGS. 23A and 23B. (A) Specificity of CHA.7.518.1.H4(S241P) towards expicells engineered to overexpress cynomolgus PVRIG and expi parentalcells. Data shows absolute geometric MFI (gMFI) measurements as afunction of increasing antibody concentration. Specificity ofCHA.7.538.1.2.H4(S241P) towards expi cells engineered to overexpresscynomolgus PVRIG and expi parental cells. Data shows absolute geometricMFI (gMFI) measurements as a function of increasing antibodyconcentration.

FIGS. 24A and 24B. (A) Blocking of PVRIG Fc to HEK cells byCHA.7.518.1.H4(S241P) and CHA.7.538.1.2.H4(S241P). Data shows thepercentage of PVRIG Fc binding to HEK cells as a function of increasingantibody concentration relative to maximum PVRIG Fc-induced signal andsecondary only background. (B) Effect of CHA.7.544 on the binding ofPVRIG Fc to HEK cells. Data shows the absolute gMFI derived from PVRIGFc binding to HEK cells in the presence of escalating concentrations ofCHA.7.544. The amount of PVRIG Fc binding was detected by an anti-mouseFc secondary conjugated to Alexa 647.

FIGS. 25A and 25B. (A) Blocking of PVRL2 Fc to HEK hPVRIG cells byCHA.7.518.1.H4(S241P), CHA.7.538.1.2.H4(S241P), and CHA.7.530.3. Datashows the percentage of PVRL2 Fc binding to HEK hPVRIG cells as afunction of increasing antibody concentration relative to maximum PVRL2Fc-induced signal and secondary only background. (B) Effect of CHA.7.544on PVRL2 Fc binding to HEK hPVRIG cells. Data shows the percentage ofPVRL2 Fc binding to HEK hPVRIG cells as a function of increasingantibody concentration relative to maximum PVRL2 Fc-induced signal andsecondary only background.

FIG. 26. Shows the percentage of Alexa 647 conjugatedCHA.7.518.1.H4(S241P) and CHA.7.538.1.2.H4(S241P) binding relative totheir maximum signal upon pre-incubation of Jurkat cells withunconjugated CHA.7.518.1.H4(S241P), CHA.7.538.1.2.H4(S241P) and anisotype control.

FIGS. 27A and 27B A) Humanized PVRIG antibodies, CHA.7.518.1.H4(S241P)and CHA.7.538.1.2.H4(S241P), increase CD4+ T cell proliferation.Representative data (n≥2) shows the percentage of CFSE low,proliferating CD4+ T cells (mean plus standard deviation) from a singlehuman CD4+ T cell donor when co-cultured with the CHO-S OKT3 hPVRL2cells in the presence of an anti-DNAM-1 antibody or different anti-PVRIGantibodies or IgG isotype controls. The dashed line indicates thebaseline percentage of CFSE low, CD4+ T cells proliferating aftertreatment with the human IgG4 isotype control antibody. The numbersrefer to the percent increase or decrease in proliferation of theanti-PVRIG or anti-DNAM-1 antibody treatments, respectively, compared tothe relevant isotype control antibodies (B) Humanized PVRIG antibodies,CHA.7.518.1.H4(S241P) and CHA.7.538.1.2.H4(S241P), increase CD4+ T cellproliferation in an hPVRL2-dependent manner. Representative data (n≥2)shows the percentage of CFSE low, proliferating CD4+ T cells (mean plusstandard deviation) from a single human CD4+ T cell donor in response toco-culture with the CHO-S OKT3 parental, or CHO-S OKT3 hPVRL2 cells inthe presence of an anti-DNAM-1 antibody or different anti-PVRIGantibodies or IgG isotype controls. The dashed line indicates thebaseline percentage of CFSE low CD4+ T cells proliferating aftertreatment with either the human IgG4 or the mouse IgG1 isotypeantibodies. The numbers refer to the percent increase or decrease inproliferation of the anti-PVRIG or anti-DNAM-1 antibody treatments,respectively, compared to the relevant isotype control antibodies.

FIG. 28A-28C. (A) Humanized PVRIG antibodies, CHA.7.518.1.H4(S241P) andCHA.7.538.1.2.H4(S241P), increase CD8+ T cell proliferation.Representative data (n≥2) shows the percentage of CFSE low,proliferating CD8+ T cells (mean plus standard deviation) from a singlehuman CD8+ T cell donor (Donor 232) when co-cultured with the CHO-S OKT3hPVRL2 cells in the presence of an anti-DNAM-1 antibody or differentanti-PVRIG antibodies or IgG isotype controls. The dashed line indicatesthe baseline percentage of CFSE low, CD8+ T cells proliferating aftertreatment with the mouse IgG1 or human IgG4 isotype antibodies. Thenumbers refer to the percent increase or decrease in proliferation ofthe anti-PVRIG or anti-DNAM-1 antibody treatments, respectively,compared to the relevant isotype control antibodies. (B) Humanized PVRIGantibodies, CHA.7.518.1.H4(S241P) and CHA.7.538.1.2.H4(S241P), increaseCD8+ T cell proliferation. Representative data (n≥2) shows thepercentage of CFSE low, proliferating CD8+ T cells (mean plus standarddeviation) from a single human CD8+ T cell donor (Donor 234) whenco-cultured with the CHO-S OKT3 hPVRL2 cells in the presence of ananti-DNAM-1 antibody or different anti-PVRIG antibodies or IgG isotypecontrols. The dashed line indicates the baseline percentage of CFSE low,CD8+ T cells proliferating after treatment with the mouse IgG1 or humanIgG4 isotype antibodies. The numbers refer to the percent increase ordecrease in proliferation of the anti-PVRIG or anti-DNAM-1 antibodytreatments, respectively, compared to the relevant isotype controlantibodies. (C) Humanized PVRIG antibodies, CHA.7.518.1.H4(S241P) andCHA.7.538.1.2.H4(S241P), increase IFNγ secretion from CD8+ T cells.Representative data (n≥2) shows the pg/ml of IFNγ produced (mean plusstandard deviation) by three different human CD8+ T cell donors (Donors231, 232, and 234) when co-cultured with the CHO-S OKT3 hPVRL2 cells inthe presence of an anti-DNAM-1 antibody or different anti-PVRIGantibodies or IgG isotype controls. The dashed line indicates thebaseline IFNγ production following treatment with the human IgG4 isotypeantibody. The numbers refer to the percent increase in IFNγ secretion ofthe anti-PVRIG antibody treatments compared to the IgG4 isotype control.

FIG. 29. Humanized PVRIG antibodies, CHA.7.518.1.H4(S241P) andCHA.7.538.1.2.H4(S241P), consistently increase CD4+ T cell proliferationacross multiple donors, while CHA.7.530.3 and CHA.7.544 do not. Thepercent proliferation relative to the isotype control was calculated bydividing the percentage of CFSE low, CD4+ T cells after PVRIG antibodytreatment over the isotype antibody treatment for each donor. Thepercent proliferation for the isotype antibody treatment was set atzero. Each symbol in the graph represents a different donor.

FIG. 30A-30D. (A) Dose-dependent effect of the humanized PVRIGantibodies, CHA.7.518.1.H4(S241P) and CHA.7.538.1.2.H4(S241P), on CD4+ Tcell proliferation. Representative data (n≥2) with 2 different humandonors shows the mean percentage of proliferating CD4+ T cells followinga dose titration of 66 nM to 0.726 nM with either the human IgG4isotype, CHA.7.518.1.H4(S241P), or CHA.7.538.1.2.H4(S241P) antibodies.The estimated EC50 is within the single digit nM range. (B)Dose-dependent effect of the humanized PVRIG antibodies,CHA.7.518.1.H4(S241P) and CHA.7.538.1.2.H4(S241P), on CD8+ T cellproliferation. Representative data (n≥2) with 2 different human donorsshows the mean percentage of proliferating CD8+ T cells following a dosetitration of 66 nM to 0.264 nM with either the human IgG4 isotype,CHA.7.518.1.H4(S241P), CHA.7.38.1.2, or CHA.7.544 antibodies. Theestimated EC50 is within the single digit nM range.

FIG. 31A-31C. (A) Flow cytometry analysis of TIGIT and PVRIG expressionon TILs and PVR, PVRL2 expression on 624 melanoma cell line. Valuesrepresent Mean fluorescent intensity (MFI) ratio vs isotype control.(B-C) Representative experiment showing IFNγ (B) and TNF (C) secretionby TILs upon co-cultured with melanoma cells 624 at 1:3 E:T for 18 hr inthe presence of isotype control, anti-TIGIT (30 μg/ml) or anti-PVRIG Abs(10 ug/ml) as mono treatment (blue histograms) or in combination withanti-TIGIT (green histograms). Percentage of Ab mono treatment effectwas compared to isotype control treatment mIgG1 and the percentage of Abcombo-treatment effect was compared to anti-TIGIT mono-treatment.

FIG. 32A-32H. TILs (209-gp100/463-F4-gp100) were co-cultured withmelanoma cells 624 in 1:3 E:T for 18 hr in the presence of anti PVRIGAbs CHA.7.518.1.H4(S241P) or CHA.7.538 with or without anti-TIGIT(aTIGIT) combo and tested for cytokine secretion. Percentage of Abtreatment effect was compared to isotype control treatment and the meanof 5 experiments (F4) or 6 experiments (209) were plotted. Paired, twotailed T test was calculated for each treatment compared to isotype orin combos-compared to anti-TIGIT alone, p values are indicated.

FIGS. 33A and 33B. (A) Humanized PVRIG antibody, CHA.7.518.1.H4(S241P),and an anti-TIGIT antibody increase CD4+ T cell proliferation comparedto single antibody treatments. Representative data (n≥2) shows thepercentage of CFSE low, proliferating CD4+ T cells (mean plus standarddeviation) from a single human CD3+ T cell donor (Donor 143) whenco-cultured with the CHO-S OKT3 hPVRL2 cells. The dashed line indicatesthe baseline percentage of CFSE low, CD4+ T cells proliferating aftertreatment with the human IgG4 isotype control antibody. (B) HumanizedPVRIG antibody, CHA.7.518.1.H4(S241P), and the anti-TIGIT antibodyincrease CD4+ T cell proliferation compared to single antibodytreatments. Representative data (n≥2) shows the percentage of CFSE low,proliferating CD4+ T cells (mean plus standard deviation) from a singlehuman CD4+ T cell donor (Donor 201) when co-cultured with the CHO-S OKT3hPVRL2 cells. The dashed line indicates the baseline percentage of CFSElow, CD4+ T cells proliferating after treatment with the human IgG4isotype control antibody. The numbers refer to the percent increase ordecrease in proliferation of the anti-PVRIG or anti-DNAM-1 antibodytreatments, respectively, compared to the relevant isotype controlantibodies.

FIGS. 34A and 34B. (A): The combination of the humanized PVRIG antibody,CHA.7.518.1.H4(S241P), and the anti-TIGIT antibody increases CD8+ T cellproliferation. Representative data (n≥2) shows the percentage of CFSElow, proliferating CD8+ T cells (mean plus standard deviation) from arepresentative human CD8+ T cell donor (Donor 232) when co-cultured withthe CHO-S OKT3 hPVRL2 cells. The dashed line indicates the baselinepercentage of CFSE low, CD8+ T cells proliferating after treatment withthe human IgG4 isotype antibody. The numbers refer to the percentincrease or decrease in proliferation of the anti-PVRIG or anti-DNAM-1antibody treatments, respectively, compared to the relevant isotypecontrol antibodies. (B) The combination of the humanized PVRIG antibody,CHA.7.518.1.H4(S241P), and the anti-TIGIT antibody increases IFNγsecretion from CD8+ T cells. Representative data (n≥2) shows the pg/mlof IFNγ produced (mean plus standard deviation) by a representativehuman CD8+ T cell donor (Donor 232) when co-cultured with the CHO-S OKT3hPVRL2 cells. The dashed line indicates the baseline IFNγ productionfollowing treatment with the human IgG4 isotype antibody. The numbersrefer to the percent increase or decrease in IFNγ secretion of theanti-PVRIG or anti-DNAM-1 antibody treatments, respectively, compared tothe relevant isotype control antibodies.

FIG. 35 depicts the design of the experimental system of Example 2(3).

FIG. 36A-36C. shows a histogram depicting levels of PVRIG (usingAnti-Human PVRIG CHA.7.538.AF647), TIGIT (using Anti-Human TIGIT Cat.17-9500-41 eBioscience) and DNAM-1 (using Anti-human CD226-APCCat.338312 biolegend) expression in TILs. Fold of expression is comparedto isotype (Iso) control.

FIG. 37. Summarized plot of the effect of anti PVRIG antibodies on thesecretion of IFNγ from TILs. TILs were co-cultured with CHO-S HLA-A2/B2Mcells over-expressing PVRL2 in E:T ratio of 1:3 for 18 hr in thepresence of anti PVRIG antibodies (c518, c538 and 544) or with antiTIGIT antibody. Each dot represents an average of data of IFNγ secretionfrom the same TIL from different experiments. The percentage indicatedis the different between each antibody treatment compared to isotypecontrol. Paired, two tailed T-test was calculated for each treatmentcompared to 544 or in combos, compared to anti TIGIT alone, p values areindicated. Number of experiments preformed per each TILs; 209 (N=3), F4(N=2), F5 (N=3) and MART1 (N=2).

FIGS. 38A and 38B. Summarized plot of the effect of c518 and c538 doseresponse on the secretion of TNF-α from TILs. TILs were co-cultured withCHO-S HLA-A2/B2M cells over-expressing PVRL2 in effector-to-target ratioof 1:3 for 18 hr in the presence of anti PVRIG antibodies (c518, c538 orisotype control) as described in Example 2(3).

FIG. 39A-39C. TILs were co-cultured with CHO-S HLA-A2/B2M target cellsover-expressing PVRL2 in E:T ration of 1:3 for 18 hr in the presence ofanti PVRIG antibodies (c518, c538 and 544) or with anti TIGIT antibody.The percentage indicated in the above tables is the difference in theeffect of cytokine secretion from TILs of each antibody treatmentcompared to its isotype control. The first experiment is represented inFigure A and B, and the second experiment in Figure C.

FIGS. 40A and 40B. CHO-S OKT3 co-culture assay design. CFSE labeled CD3+T cells were co-cultured with CHO-S-OKT3-PVRL2 or mock transfected cellsfor 5 d. The effect anti-PVRIG Abs on T cell proliferation and cytokinesecretion was analyzed.

FIGS. 41A and 41B. Effect of anti-PVRIG antibodies on IFNγ secretionupon CHO-OKT3 PVRL2 cells in responder vs. non-responder donor. CD3+cells from 2 different donors were co-cultured with CHO-S-PVRL2 cells in5:1 E:T for 5 d in the presence of anti PVRIG Abs and tested forcytokine secretion and T cells proliferation. (A) ‘responder donor’ inwhich we observed an effect to anti PVRIG Abs. (B) ‘non-responder donor’in which we do not observed effects to Abs treatment.

FIGS. 42A and 42B. Effect of anti-PVRIG antibodies on CD4 and CD8proliferation from responder donor. CFSE labeled CD3+ T cells wereco-cultured with CHO-S-PVRL2 cells in 5:1 E:T for 5 d in the presence ofanti PVRIG Abs or anti-TIGIT Abs. The effect on T cells proliferationgating on CD4 or CD8 was evaluated by flow cytometry. Percentage ofproliferating cells (CFSE low) (A) or total cells number (B) ofCD4+CFSElow or CD8+CFSE low are presented.

FIG. 43A-43C. Shows the effect of anti-PVRIG antibodies on IFNγsecretion or CD8 proliferation from responder donor. CD3+ cells wereco-cultured with CHO-S-PVRL2 cells in 5:1 E:T for 5 d in the presence ofanti PVRIG Abs and tested for (A) cytokine secretion and (B) T cellsproliferation. Percentage of Ab treatment effect was compared to isotypecontrol treatment and the mean of 5 ‘responders’ donors (responders) ispresented. (C) IFNγ secretion levels from the same 5 donors uponco-culture with CHOS-OKT3 PVRL2 as described in section A and B upontreatment with isotype vs. anti-PVRIG Abs. p value represent ratiopaired T test.

FIG. 44 is a summary table of Abs treatment effect across donors tested(n=10). Percentages indicated represent the effect of Ab treatment on aspecific readout (indicated in columns titles) as compared to therelevant isotype control. ‘responder’ donors (donors #3, 72,226,345 andES_001) considered as ‘responder’ which some anti-PVRIG Abs (mainlyCHA.7.518) enhanced IFNγ or proliferation vs. isotype controls.

FIG. 45A-45C depict the results of experiments with several antibodies.The affinities (nM) are shown in A, with the HEK hPVRIG cells being HEKcells transformed with hPVRIG as discussed herein and Jurkat cellsexpressing endogeneous hPVRIG. (B) depicts the gMFI using 4 differentantibodies against Donor 1 primary CD8 T cells and (C) being Donor 2primary CD8 T cells.

FIGS. 46A and 46B depict interactions of TIGIT with CHO cells. (A) HumanTIGIT Fc protein binds to CHO cells. Graded concentrations of humanTIGIT Fc and synagis IgG1 control were assessed for their ability tobind to CHO cells in a FACS-based binding assay. (B) Human PVR isexpressed on activated CD4 T cells. CD4 T cells were co-cultured withCHO cells expressing the scFv of the OKT3 antibody and activated for 5days. On day 5, CD4 T cells were analysed for expression of PVR anddilution of CFSE.

FIG. 47A-47C depict antitumor responses of anti-mPVRIg and anti-PDL-1antibodies in CT26 tumor model. A-B. Groups of 10 BALB/c mice weresubcutaneously injected with 5×10⁵ CT26 cells. After tumors weremeasured on day 4, mice were randomized (40 mm3 mean tumor volume pergroup) and then treated with the designated mAb (100 or 200 μg/dose IP)followed by additional doses on days 7, 11, 14, 18 and 21. A. Groupswere treated with 6 doses of single agents. Anti-PDL-1 vs control***p<0.0001. Tumor volumes are represented as the Mean volume+SEM. B.Tumor volumes were measured twice weekly. The number of tumor-free (TF)mice per group is indicated. C. survival proportions of assigned groups;Anti-PDL-1 vs control **p=0.005.

FIG. 48A-48C depict antitumor responses of anti-PVRIG and anti-PDL-1antibodies combination in CT26 tumor model. A-B. Groups of 10 BALB/cmice were subcutaneously injected with 5×10⁵ CT26 cells. After tumorswere measured on day 7, mice were randomized (75 mm³ mean tumor volumeper group) and then treated with the designated mAb (300 μg/dose IP)followed by additional doses on days 11, 14, 18, 21 and 25. A. Groupswere treated with 6 doses of combined agents. Anti-PDL-1+mAb 407 vscontrol p=0.0005; anti-PDL-1 and mAb 406 vs control p=0.056. B. Tumorvolumes were measured ×3 weekly. The number of tumor-free (TF) mice pergroup is indicated. C. survival proportions of assigned groups;Anti-PDL-1+mAb 407 vs control *p=0.0088.

FIG. 49A-49D depict the amino acid sequences and the nucleic acidsequence for the variable heavy chain (A and B, respectfully) and theamino acid sequences and the nucleic acid sequence for the variablelight chain (C and D, respectfully) for AB-407 (BOJ-5G4-F4).

FIGS. 50A and 50B depicts the amino acid sequences of the constantdomains of human IgG1 (with some useful amino acid substitutions), IgG2,IgG3, IgG4, IgG4 with a hinge variant that finds particular use in thepresent invention, and the constant domains of the kappa and lambdalight chains.

FIG. 51 depicts the sequences of human and cynomolgus macaque (referredto as cyno) TIGIT ECD and of the human PVR ECD proteins.

FIG. 52. Shows the flow cytometry binding summary for anti-TIGIT fabs.All unique ELISA positive fabs were analyzed by flow cytometry. The meanfluorescence intensity (MFI) was measured for the human or cyno TIGITover-expressing Expi293 cells as well as the parental Expi293 cells. TheMFI ratio for the target-specific vs off-target binding was calculated.Data for selected clones is shown.

FIG. 53A-53C depict the sequences of anti-TIGIT antibodies. Unlessotherwise noted, the CDRs utilize the IMGT numbering (including theantibodies of the sequence listing.

FIG. 54. Shows the FACS KD results of anti-TIGIT mAbs binding to Expi293human TIGIT over-expressing cells as described in Example 12.

FIG. 55. Shows the FACS KD results of mAbs binding to Expi293 cyno TIGITover-expressing cells.

FIG. 56. Shows the results from Example 14, showing the resultingkinetic rate constants and the equilibrium dissociation constants wheredata were reliable enough to estimate the binding constants.

FIGS. 57A and 57B show the results of human PVR-Fc variant binding toExpi293 human TIGIT over-expressing cells in Example 4. Figure A (left):Binding curve generated for human PVR-m2aFc construct titrated withExpi293 human TIGT over-expressing cells. The KD and 95% confidenceinterval are shown. Figure B (right): Binding curve generated for humanPVR-h1Fc construct titrated with Expi293 human TIGT over-expressingcells. The KD and 95% confidence interval are shown.

FIG. 58. Shows a table of phage antibodies inhibiting human PVR-m2aFcbinding to human TIGIT over-expressed on Expi293 cells. mAbs were testedagainst known blocking (BM26) benchmark antibody, and human IgG4 isotypecontrol (Synagis) antibody. A “Yes” indicates the mAb inhibited hPVRanalogous to BM26.

FIG. 59. Shows a table of IC50 values of anti-TIGIT hybridoma antibodiesinhibiting binding of human PVR-h1Fc to human TIGIT over-expressed onExpi293 cells. Values are representative of one of two independentexperiments. The IC50 results for the two independently performedexperiments showed a range of only 1.2-2-fold differences.

FIG. 60. Shows the results of Example 6, that the phage-derived and BManti-human TIGIT antibodies, CPA.9.027, CPA.9.049, CPA.9.059, BM26, andBM29 increase IL-2 signaling. BM26 and BM29 are both the human IgG4(hIgG4 with a S241P variant) isotype. Representative data (n≥2) showsthe RLU (mean+/−standard deviation) of the luciferase signal from a 6hour co-culture of Jurkat IL-2-RE luciferase human TIGIT cells and aAPCCHO-K1 human PVR cells. The concentration of each antibody was 10 μg/ml.

FIG. 61. Shows additional results of Example 6, that the phage-derivedand BM hIgG4 anti-human TIGIT antibodies, CPA.9.027, CPA.9.049,CPA.9.059, BM26, and BM29 increase IL-2 signaling in a dose-dependentmanner. BM26 and BM29 are both the hIgG4 isotype. Representative data(n≥2) shows the RLU (mean+/−standard deviation) of the luciferase signalfrom a 6 hour co-culture of Jurkat IL-2-RE luciferase human TIGIT cellsand aAPC CHO-K1 human PVR cells. A 10 point, 2-fold dilution seriesstarting at 20 μg/ml was used for each antibody.

FIG. 62. Shows the results of Example 6, that the hybridoma-derived andBM anti-human TIGIT antibodies, CHA.9.536, CHA.9.541, CHA.9.546,CHA.9.547, CHA.9.560, BM26, and BM29 increase IL-2 signaling. BM26 andBM29 are both the mIgG1 isotype. The non-blocking anti-human TIGITantibody, CHA.9.543 does not enhance IL-2 signaling. Representative data(n≥2) shows the RLU (mean+/−standard deviation) of the luciferase signalfrom a 6 hour co-culture of Jurkat IL-2-RE luciferase human TIGIT cellsand aAPC CHO-K1 human PVR cells. The concentration of each antibody was10 μg/ml.

FIG. 63. Shows the results of Example 6, that the hybridoma-derived andbenchmark mIgG1 anti-human TIGIT antibodies, CHA.9.536, CHA.9.541,CHA.9.546, CHA.9.547, CHA.9.560, and BM26 increase IL-2 signaling in adose-dependent manner. BM26 is the mIgG1 isotype. Representative data(n≥2) shows the RLU (mean+/−standard deviation) of the luciferase signalfrom a 6 hour co-culture of Jurkat IL-2-RE luciferase human TIGIT cellsand aAPC CHO-K1 human PVR cells. A 10 point, 2-fold dilution seriesstarting at 20 μg/ml was used for each antibody.

FIG. 64. Shows that the phage, hybridoma and BM anti-human TIGITantibodies, CPA.9.027, CPA.9.049, CPA.9.059, CHA.9.536, CHA.9.541,CHA.9.546, CHA.9.547, CHA.9.560, BM26, and BM29 increaseantigen-specific IFNγ signaling. BM26 is tested as both the hIgG4 andmIgG1 isotypes, while BM29 is only tested as the hIgG4 isotype.Representative data (n=2) shows the amount of IFNγ (mean+/−standarddeviation) in the culture supernatant after 24-hour co-culture ofCMV-specific CD8⁺ T cells with the Mel624 human PVR cells. Theconcentration of each antibody was 10 μg/ml. The Mel624 human PVR usedin the assay were pulsed with 0.0033 μg/ml or 0.001 μg/ml peptide.

FIG. 65. Shows that the phage, hybridoma and BM anti-human TIGITantibodies, CPA.9.027, CPA.9.049, CPA.9.059, CHA.9.536, CHA.9.541,CHA.9.546, CHA.9.547, and CHA.9.560, as well as BM26, increaseantigen-specific IFNγ signaling either alone (open bars) or incombination with an anti-PVRIG antibody, CHA.7.518.1.H4(S241P) (hatchedbars). BM26 is the mIgG1 isotype. For the isotype antibody controltreatments, the open bar refers to the isotype antibody alone, and thehatched bar refers to isotype antibody in combination withCHA.7.518.1.H4(S241P). Representative data (n=2) shows the amount ofIFNγ (mean+/−standard deviation) in the culture supernatant after a 24hour co-culture of CMV-specific CD8+ T cells with Mel624 cellsover-expressing human PVR and human PVRL2. The concentration of eachantibody was 10 μg/ml. The Mel624 human PVR/human PVRL2 cells used inthe assay were pulsed with 0.0033 μg/ml or 0.001 μg/ml peptide.

FIG. 66. Shows the percent increase of IFNγ secretion with anti-humanTIGIT antibodies, CHA.7.518.1.H4(S241P), and the combination ofanti-human TIGIT antibodies and CHA.7.518.1.H4(S241P), over therespective isotype control antibodies.

FIG. 67 is the dendrogram for the epitope binning experiments of Example7.

FIG. 68 is the grouping of the antibodies from the epitope binningexperiments of Example 7.

FIG. 69. Shows the high affinity binding to human TIGIT overexpressingcells in a dose titration of the affinity matured phage antibodies(CPA.9.083, CPA.9.086), humanized hybridoma antibodies (CHA.9.547.7,CHA.9.547.13), benchmark antibodies (BM26, BM29), and the hIgG4 isotypecontrol (anti-Synagis) on human TIGIT over-expressing Expi293 cells, asdescribed in experiments of Example 3. All antibodies were titratedusing a serial 2-fold dilution over 11 points starting at 10 μg/ml(133.33 nM [binding site]). AF647-labeled goat anti-human F(ab′)(Jackson Immunoresearch) was added to the cells to detect binding ofanti-TIGIT antibodies. The gMFI of the anti-TIGIT antibodies bound tothe human TIGIT over-expressing Expi293 cells (black line), and theparental Expi293 cells (grey line) are shown. K_(D) values+/−95% CI, andcurve fits are indicated below each graph.

FIG. 70. Shows that anti-TIGIT antibodies are cross reactive to cynoTIGIT in a dose titration of the affinity matured phage antibodies(CPA.9.083, CPA.9.086), humanized hybridoma antibodies (CHA.9.547.7,CHA.9.547.13), benchmark antibodies (BM26, BM29), and the hIgG4 isotypecontrol (anti-Synagis) on cyno TIGIT over-expressing Expi293 cells, asdescribed in experiments of Example 3. All antibodies were titratedusing a serial 2-fold dilution over 11 points starting at 10 μg/ml(133.33 nM [binding site]). AF647-labeled goat anti-human F(ab′)(Jackson Immunoresearch) was added to the cells to detect binding ofanti-TIGIT antibodies. The gMFI of the anti-TIGIT antibodies bound tothe cyno TIGIT over-expressing Expi293 cells (black line), and theparental Expi293 cells (grey line) are shown. K_(D) values+/−95% CI, andcurve fits are indicated below each graph.

FIGS. 71A and 71B. Shows that affinity matured phage antibodies arecross reactive to mouse TIGIT in a dose titration of the affinitymatured phage antibodies reformatted as mouse IgG1 (mIgG1) (CPA.9.083,CPA.9.086), benchmark anti-mouse TIGIT antibodies (BM27 mIgG1, BM30mIgG1), and the mIgG1 isotype control (anti-Synagis) are shown, asdescribed in experiments of Example 3. A) The gMFI of the anti-TIGITantibodies bound to the mouse TIGIT over-expressing HEK cells (blackline), and the parental HEK cells (grey line). B) The gMFI of theanti-TIGIT antibodies (black line) or Synagis mIgG1 (grey line) bound toregulatory CD4+CD25+Foxp3+ T cells isolated from s.c. implanted Rencatumors in Balb/c mice. Anti-TIGIT antibodies were titrated using eithera serial 2- or 3-fold dilution series starting at 15 μg/ml (200 nM[binding site]), or 10 μg/ml (132 nM [binding site]), respectively.AF647-labeled goat anti-mouse IgG-Fc (Southern Biotech) were added tothe cells to detect binding of the anti-TIGIT antibodies on mouse TIGITover-expressing cells. Anti-TIGIT antibodies were directly conjugated toAF647 for mouse Treg binding. K_(D) values for each anti-TIGIT antibodyare indicated.

FIG. 72. Shows a dose titration of the affinity matured phage antibodies(CPA.9.083, CPA.9.086), humanized hybridoma antibodies (CHA.9.547.7,CHA.9.547.13), and benchmark antibodies (BM26, BM29) on human effectormemory CD95+CD28−CD8+CD3+ T cells from 3 healthy donor PBMCs (Donors321, 322, and 334), as described in experiments of Example 3. PBMCs weresurface stained with antibodies against the following lineage markersCD3, CD4, CD8, CD14, CD16, CD28, CD56, and CD95 (BD Biosciences,BioLegend), as well as live/dead fixable aqua dye (Life Technologies).AF647-labeled anti-TIGIT antibodies and hIgG4 isotype control antibody(anti-Synagis) were then titrated using a serial 3-fold dilution over 12points starting at 30 μg/ml (396 nM [binding site]). The gMFI of theanti-TIGIT antibodies bound to the effector memory T cells are shown. KDvalues for each antibody across the 3 different donors are reported inthe table. The affinity mature phage antibodies (CPA.9.083 andCPA.9.086) had the highest binding affinity to the human effector memoryT cells.

FIG. 73. Shows a dose titration of the affinity matured phage antibodies(CPA.9.083, CPA.9.086, CPA.9.103), humanized hybridoma antibody(CHA.9.547.1), and benchmark antibody (BM26) on cyno effector memoryCD95⁺CD28⁻CD8⁺CD3⁺ T cells from PBMCs isolated from 2 naïve cyno monkeys(BioreclamationIVT), as described in experiments of Example 3. PBMCswere surface stained with antibodies against the following lineagemarkers CD3, CD4, CD8, CD14, CD16, CD28, CD56, and CD95 (BD Biosciences,BioLegend), as well as live/dead fixable aqua dye (Life Technologies).AF647-labeled anti-TIGIT antibodies and hIgG4 isotype control antibody(anti-Synagis) were then titrated using a serial 3-fold dilution over 12points starting at 30 μg/ml (396 nM [binding site]). The gMFI of theanti-TIGIT antibodies bound to the effector memory T cells are shownwith the gMFI of the anti-Synagis hIgG4 isotype control antibodysubtracted. K_(D) values for each antibody across the 2 donors arereported in the table. The affinity mature phage antibodies (CPA.9.083and CPA.9.086) had the highest binding affinity to the cyno effectormemory T cells.

FIG. 74. Shows the SPR kinetics of anti-TIGIT antibody binding to human,cyno and mouse TIGIT, as described in experiments of Example 5. Thekinetic rate and equilibrium dissociation constants for the affinitymatured phage antibodies (CPA.9.083, CPA.9.086, CPA.9.103), humanizedhybridoma antibodies (CHA.9.547.1 and CHA.9.547.7), and benchmarkantibodies (BM26, BM29) were determined by SPR on the ProteOninstrument.

FIG. 75 shows that the anti-TIGIT antibodies block PVR/TIGITinteractions, as described in experiments of Example 4. Human TIGITover-expressing Expi293 cells were preincubated with either the affinitymatured phage antibodies (CPA.9.083, CPA.9.086), humanized hybridomaantibodies (CHA.9.547.7, CHA.9.547.13), benchmark antibodies (BM26,BM29), or the hIgG4 isotype control (anti-Synagis). All antibodies weretitrated using a serial 2.5-fold dilution over 11 points starting at 10μg/ml (133.33 nM [binding site]). Following antibody preincubation,human PVR-m2aFc was added to the cells at 158 nM [binding site] or EC₉₀.AF647-labeled goat anti-mouse IgG-Fc (Southern Biotech) was then addedto the cells to detect binding of anti-TIGIT antibodies. The percentinhibition of PVR-m2aFc binding to the human TIGIT over-expressingExpi293 cells is shown for each antibody. IC₅₀ values for eachanti-human TIGIT antibody are reported in the table (n=2 experiments).

FIG. 76. Show the results of Example 6, that the affinity matured phageantibodies (CPA.9.083, CPA.9.086), humanized hybridoma antibodies(CHA.9.547.7, CHA.9.547.13), and benchmark antibody (BM26) increase IL-2signaling in a dose-dependent manner. Synagis hIgG4 is the isotypecontrol antibody. Representative data (n≥2) shows the RLU(mean+/−standard deviation) of the luciferase signal from a 6 hourco-culture of Jurkat IL-2-RE luciferase human TIGIT cells and CHO-K1human PVR cells. A 19 point, 1.5-fold dilution series starting at 20μg/ml was used for each antibody.

FIG. 77. Shows that anti-TIGIT antibodies induce IFNγ in CMV-specificCD8+ T cells. An in vitro co-culture assay with human CMV-specific CD8+T cells was utilized to assess the effect of the affinity matured phageantibodies (CPA.9.083, CPA.9.086), humanized hybridoma antibodies(CHA.9.547.7, CHA.9.547.13), and benchmark antibodies (BM26, BM29) onantigen-specific cytokine secretion, as described in experiments ofExample 6. The target cell line used in the assay was the HLA-A2+pancreatic adenocarcinoma cells, Panc.05.04 that endogenously expresseshuman PVR and PVRL2. Panc.05.04 cells were pulsed with the CMV pp65peptide at 0.03 μg/ml or 0.01 μg/ml at 37° C. for 1 hour. Cells werethen washed and plated at 50,000 cells/well in 96-well round-bottomtissue culture treated plates. Anti-human TIGIT antibodies or theisotype control hIgG4 antibody (anti-Synagis) were added at aconcentration of 0.1 μg/ml. Human CMV-specific CD8+ T cells from asingle donor were expanded according to the protocol above. 50,000 humanCD8+ T cells were added to each well. Co-cultures were incubated at 37°C. with 5% CO2 for 24 hours. The amount of human interferon gamma (IFNγ)in the co-culture supernatant was measured by flow cytometry using acytometric bead assay (BD Biosciences). The percent increase of IFNγsecretion for each antibody over the hIgG4 isotype is summarized in thetable (n=2 experiments).

FIG. 78. Shows anti-TIGIT antibodies augment IFNγ when combined with aPVRIG antibody, CHA.7.518.1.H4(S241P). An in vitro co-culture assay withhuman CMV-specific CD8+ T cells was utilized to assess the effect of theaffinity matured phage antibodies (CPA.9.083, CPA.9.086), humanizedhybridoma antibodies (CHA.9.547.7, CHA.9.547.13), and benchmarkantibodies (BM26, BM29) on antigen-specific cytokine secretion incombination with an anti-PVRIG antibody, CHA.7.518.1. The target cellline used in the assay was the HLA-A2+ pancreatic adenocarcinoma cells,Panc.05.04 that endogenously expresses human PVR and PVRL2. Panc.05.04cells were pulsed with the CMV pp65 peptide at 0.03 μg/ml or 0.01 μg/mlat 37° C. for 1 hour. Cells were then washed and plated at 50,000cells/well in 96-well round-bottom tissue culture treated plates.Anti-human TIGIT antibodies or the isotype control hIgG4 antibody(anti-Synagis) were added at a concentration of 0.1 μg/ml in combinationwith CHA.7.518.1 (hatched bars) or a control hIgG4 isotype antibody at10 μg/ml (solid bars). Human CMV-specific CD8+ T cells from a singledonor were expanded according to the protocol above. 50,000 human CD8+ Tcells were added to each well. Co-cultures were incubated at 37° C. with5% CO2 for 24 hours. The amount of human IFNγ in the co-culturesupernatant was measured by flow cytometry using a cytometric bead assay(BD Biosciences). The percent increase of IFNγ secretion for eachantibody over the hIgG4 isotype is summarized in the table (n=2experiments).

FIG. 79. Shows the correlation analysis of PVRIG and TIGIT expression onCD4+ and CD8+ T cells from dissociated tumors. For each tumor sample, amean flourescence intensity ratio (MFIr) was calculated, and aSpearman's correlation analysis was performed, and an r2 and p valuereported.

FIGS. 80A-80C. Shows the results of tumor growth inhibition and survivalin TIGIT KO mice treated with an anti-mouse PVRIG antibody. Groups of7-10 TIGIT KO and C57BL/6 WT mice were s.c. injected with1×10⁵B16/Db-hmgp100 cells. Mice were treated twice per week for 3 weeks,starting at the inoculation day (day 0) with the designated antibody. A)Mean tumor volumes+/−standard error of the mean (SEM) are shown in theupper graph, with *** indicating a p-value<0.001 for TIGIT KO treatedwith anti-mouse PVRIG antibody (Clone 407) compared to C57BL/6 WTtreated with the mIgG1 isotype control antibody. Tumor volumes forindividual mice within each antibody treatment group are shown as spiderplots in lower graphs. B) Table summarizing the TGI as measured atindicated days compared to control C57BL/6 WT mice treated with themIgG1 isotype control. C) Survival of mice after s.c. injection ofB16/Db-hmgp100 cells.

FIG. 81 depicts combination treatments with the indicated antibodies ascompared to control in Mel-624, Colo205, and Panc.05.04 cells. gp100 orCMVpp65 specific T cells were co-cultured with Mel-624, Colo205, andPanc.05.04 cells, gp100 or CMVpp65 peptide, and the indicated antibodiesat 10 mg/ml. IFN-γ concentration in the conditioned media was determinedat 24 hrs. Average+Std Dev of triplicates is shown. % change in IFN-γfor each condition relative to hIgG4 is shown.

FIG. 82A-82C depict expression of PD-1/TIGIT/PVRIG on CD8 T cells andexpression of PD-L1, PVR, PVRL2 on Colo205, Panc.05.04 cells. A)Expression of PVRIG, TIGIT, and PD-1 on CMVpp65 reactive T cellsexpanded with pp65 peptide with IL-2 and IL-7 for 10 days. Expression ofPVRIG, TIGIT, and PD-1 on CMVpp65 reactive T cells is shown. B)Expression of PD-L1, PVR, and PVRL2 on Colo205 and Panc.05.04 cells isshown. C) CMVpp65 specific T cells were co-cultured with Colo205 andPanc.05.04 cells, CMVpp65 peptide, and the indicated antibodies at 10mg/ml. IFN-γ concentration in the conditioned media was determined at 24hrs. Average+Std Dev of triplicates is shown. % change in IFN-γ for eachcondition relative to hIgG4 is shown.

FIG. 83A-83H. PVRIG is expressed highest on cytotoxic lymphocyte subsetsfrom human cancer. A) Expression of PVRIG on leukocyte cell subsets from5-8 healthy donor PBMCs is shown. PVRIG expression is defined as theratio of PVRIG MFI relative to isotype control MFI. B) Expression ofPVRIG, TIGIT, CD96, and PD-1 on peripheral blood Tregs as compared toCD8 T cell subsets from 5 healthy donor PBMCs is shown. C) CMV pp65specific T cells from 3 healthy donors were expanded in vitro with pp65(495-503) peptide, IL-2 and IL-7 for up to 7 days. Expression of TIGIT(blue) and PVRIG (black) on HLA-A2/pp65 (495-503) tetramer positivecells is shown. D) Human T cells were cultured with allogeneic DCs andexpression of TIGIT and PVRIG shown on CD4⁺ T cells on day 0, 1, 2, and7 post activation. E) Representative FACS plots showing expression ofPVRIG (blue) compared to isotype control (red) on TILS (CD4 T cells, CD8T cells, and NK cells) from a representative lung and kidney cancer. F)Co-expression of PVRIG, TIGIT, and PD-1 on CD4 and CD8 TILS from a lungcancer sample is shown. G) Expression of PVRIG on CD8⁺ and CD4⁺ TILSfrom dissociated human tumors of various cancer types is shown. Each dotrepresents a distinct tumor from an individual patient. H) Relativeexpression on CD8 TILs vs Treg TILS for PVRIG, TIGIT, and PD-1 fromendometrial, kidney, and lung tumors was assessed. For each tumor, thefold expression on CD8 TILS was normalized to fold expression on TregTILS and plotted. For A, B, C, G, and H, mean±SEM is shown by the errorbars.

FIG. 84A-84F. PVRL2 expression is enhanced in the tumormicroenvironment. A) PVRL2 expression was assessed by IHC on lung,ovarian/endometrial, breast, colon, kidney, and melanoma tumors. Barsdepict mean±SEM. For each tumor, 2 cores were assessed by a pathologistand scored based on prevalence and intensity of membranous staining ontumor cells as described in the supplemental methods. For each tumor,the average score of 2 cores is shown. B) A representative melanomatumor showing PVRL2 expression on tumor cells (arrow) and in the immunecells (*) in the stroma is shown. C) PVRL2 expression on a log 2 scalefrom dissociated tumors determined by FACS on CD45⁻, CD14⁺ TAMs, andLin⁻CD14⁻CD33^(hi) mDC cell subsets is shown. Mean±SEM is shown for eachcancer type. Dotted line represents no staining was observed. For eachcell type, at least 100 events were required in order to be analyzed. D)Representative FACS plots for PVRL2 expression (blue) as compared to IgG(red) are shown for a lung cancer. E) For tumor samples where we wereable to assess both PVRIG and PVRL2 expression, PVRIG expression on CD8⁺T cells is plotted versus PVRL2 expression on CD14⁺ TAMS and CD45⁻ cellsfor each tumor. Each dot represents an individual tumor sample. Red linerepresents a 2 fold expression of PVRIG or PVRL2 compared to IgG. TheTable in FIG. 84F shows the prevalence of PVRL2 in various tumorsamples.

FIG. 85A-85E. Distinct regulation of PVRL2 and PD-L1 on tumor cells. A)Expression of PD-L1 and PVRL2 was assessed by IHC on serial sections.Tumors samples from FIG. 84A were grouped based on tissue type andexpression of PVRL2 on PD-L1 negative and PD-L1 positive is shown. PD-L1negative tumors were defined as no membranous staining on tumor orimmune cells from either duplicate cores for a given tumor. PD-L1positive staining was defined as membranous staining on at least 1 coreof a tumor. Bars depict mean±SEM for each group. B, C) Representativeexpression of a PVRL2_(+PD-L)1⁻ endometrial (B) tumor and a PVRL2⁺PD-L1⁻lung (C) tumor. D) Immature BM-DCs were cultured with the indicatedstimuli and PVR, PVRL2, and PD-L1 expression assessed by FACS on day 2of culture. For each condition, expression was normalized to media onlycontrol condition. E) Expression of PVR, PVRL2, and PD-L1 on HT-29 cellstreated with IFN-γ or media alone is shown. PD-L1 or PVRL2 is shown inblue and IgG isotype control staining is shown in red.

FIG. 86A-86I. CHA.7.518.1.H4(S241P) is a high affinity antibody thatenhances T cell activation. A) Binding of CHA.7.518.1.H4(S241P) or IgGisotype control to HEK293 PVRIG or HEK293 parental cells by FACS isshown. FACS KD values are shown for the binding of CHA.7.518.1.H4(S241P)to HEK293 hPVRIG, HEK293 cPVRIG, and Jurkat cells. B)CHA.7.518.1.H4(S241P) disrupts the binding of PVRL2 Fc to HEK293 cellsectopically expressing PVRIG. Mean±Std Dev of triplicate values isshown. C) CHA.7.518.1.H4(S241P) blocks the binding of PVRIG Fc to HEK293cells that endogenously express PVRL2. D) Human CD4 T cells wereco-cultured with aAPC CHO cells expressing a cell surface bound anti-CD3antibody and hPVRL2 in the presence of 10 μg/ml anti-PVRIG antibody andhuman IgG isotype control antibodies. The effect of anti-PVRIG Ab onproliferation of CD4 T cells isolated from 11 different donors is shown.Bars depicted mean±SEM. E) gp100 specific T cell lines (TIL-209,TIL-463) were co-cultured with CHO cells engineered to express HLA-A2and PVRL2 along with 10 μg/ml anti-PVRIG or IgG isotype controlantibody. IFN-γ and TNF-α production was tested at 24 hours postco-culture. Mean±Std Dev of triplicate values is shown. Percent changein IFN-γ and TNF-α for each condition relative to isotype control isdepicted by the number above each bar F) Expression of PVR, PVRL2, andPD-L1 (red) relative to IgG (blue) on MEL624, Colo205, and Panc.05.04cells is shown. For the T cells, expression of PVRIG, TIGIT, and PD-1(red) relative to IgG (blue) on TIL-209 and TIL-463 gp100 specific Tcells, and on CMVpp65 specific T cells is shown. To expand CMVpp65reactive T cells, PBMCs were cultured with pp65 (495-503) peptide, IL-2,and IL-7 for 10 days. Expression of PVRIG, TIGIT, PD-1 is shown onHLA-A2/pp65 tetramer positive cells. G) gp100 specific T cells (TIL-209,TIL-463) expanded from TILS derived from melanoma tumors wereco-cultured with MEL624 cells in the presence of 10 μg/ml of theindicated antibodies. IFN-γ concentration in the conditioned media wasdetermined at 24 hrs. H, I) Expanded CMVpp65 specific T cells wereco-cultured with Colo205 and Panc.05.04 cells, CMVpp65 peptide, and theindicated antibodies at 10 μg/ml. IFN-γ concentration in the conditionedmedia was determined at 24 hrs. For E, G, H, I, average±Std Dev oftriplicates is shown. Percent change in IFN-γ for each conditionrelative to isotype control is depicted by the number above each bar.

FIG. 87A-87E. PVRIG deficient mice have increased T cell function. A)RNA expression of PVRIG as measured by qRT-PCR from purified mouseimmune cell subsets was assessed. Relative expression to housekeepingwas determined by □Ct method. B) pmel CD8⁺ TCR transgenic T cells wereactivated with gp100 (25-33) and PVRIG and TIGIT RNA transcript levelsassessed by qRT-PCR at the indicated time points. Graph shows mean±SEMof results from 5 different experiments. C) Spleens were harvested fromPVRIG^(−/−) and WT littermates and analyzed by flow cytometry forexpression of PVRIG on NK, CD4⁺ and CD8⁺ T cells (“Resting” cells). Inaddition, CD3⁺ T cells were isolated from splenocytes and activated for11 days with anti-CD3/anti-CD28 beads. Following the activation, PVRIGexpression on CD4⁺ and CD8⁺ T cells (“activated” cells) was analyzed byflow cytometry. Each dot represents cells derived from an individualmouse. D) WT and PVRIG^(−/−) derived splenocytes were labeled with CellProliferation Dye eFluor450 and were cultured in the presence ofControl-Fc (mouse IgG2a) or with mouse PVRL2 Fc. After 4 d of culture,cell division was analyzed by flow cytometry. Representative FACS plotsfrom an experiment (left) and the summary of percentage inhibition byPVRL2 Fc (defined as % proliferation Control-Fc subtracted from %proliferation PVRL2 Fc) 3 independent experiments (right) arepresented. * indicate p-value<0.05, paired student's t-test for thechange in proliferation in the presence of PVRL2-FC relative toproliferation in the presence of protein control in WT versusPVRIG^(−/−) T cells. E) pmel CD8⁺ T cells derived from pmel PVRIG^(−/−)or pmel PVRIG WT mice were activated for 11 days with their cognatepeptide and IL2. Activated pmel CD8⁺ cells were then co-cultured withB16-Db/gp100 cells for 18 hours and following the co-culture wereevaluated for CD107 expression and for cytokine production. Fourindependent experiments are presented as indicated by each paired dot. *indicate p-value<0.05, Student's t-test comparing PVRIG^(−/−) versus WT.

FIG. 88A-88H. PVRIG deficiency results in reduced tumor growth andincreased CD8+ effector T cell mechanism. A) C57BL/6 WT or PVRIG^(−/−)mice were subcutaneously injected with 5×10⁵ MC38 cells. Tumor volumeswere measured ×2 weekly. n=10 mice per group, Ave±SEM is shown, *Indicate p-value<0.05 by Student's unpaired t-test for WT mice versusPVRIG^(−/−) mice (ANOVA). B) Individual tumor growth curves are shown.n=10 mice per group, one representative experiment is shown (n=2). C)C57BL/6 WT or PVRIG^(−/−) mice were subcutaneously injected with 5×10⁵MC38 cells. At day 14 post-inoculation, mice were treated withanti-PD-L1, ×2 weekly for 2 weeks. Tumor volumes were measured ×2weekly. n=10 mice per group, Ave±SEM is shown, p-value=0.052 byStudent's unpaired t-test for WT mice versus PVRIG^(−/−) mice, bothtreated with anti-PD-L1. D) Individual tumor growth curves are shown.One representative experiment is shown (n=2). E-H) In separate duplicateexperiments, tumors were harvested on day 18 after mice had received 2doses of anti-PD-L1 or the relevant isotype control. Dissociated tumorswere enriched for CD45⁺ cells prior to stimulation for 4 hours with PMAand Ionomycin in the presence of Brefeldin A. Graphs illustrate thetotal numbers per mg tumor tissue of CD45⁺ immune cells, CD8⁺ T cellsand Interferon-γ-producing CD8⁺ T cells from isotype-treated wild-typeand PVRIG^(−/−) mice (E) and from anti-PD-L1-treated wild-type andPVRIG^(−/−) mice (F). G-H) Frequency of CD8⁺ IFN-γ⁺ TNF-α⁺ effectorcells in tumor-draining lymph nodes from isotype- and anti-PD-L1-treatedPVRIG^(−/−) mice, relative to their corresponding wild-type cohort isshown. For E-H, Ave±SEM is shown and p values from a Student's unpairedt-test is shown.

FIG. 89A-89F. Antagonistic anti-PVRIG antibodies synergistically inhibittumor grown in combination of PD-1 inhibitors or TIGIT geneticdeficiency. A) Binding of mPVRL2 Fc fusion protein to mPVRIG HEK293engineered cells that were pre-incubated with serial dilutions ofanti-mPVRIG mAb or IgG isotype control Ab is shown. B) BALB/c mice weresubcutaneously injected with 5×10⁵ CT26 cells. On day 14 postinoculation, mice were sacrificed and spleen, draining lymph nodes andtumors were harvested. Cells were analyzed by flow cytometry forexpression of PVRIG on CD3⁺CD4+ T cells, CD3⁺CD8⁺ T cells, CD3⁻CD49b⁺ NKcells, CD11b⁺ Gr-1⁺ Myeloid-Derived-Suppressor Cells (MDSC) andCD11b⁺F4/80⁺ macrophages. C,D) BALB/c mice were subcutaneously injectedwith 5×10⁵ CT26 cells. At day 7 post inoculation mice were treated withanti-PD-L1 and/or anti-PVRIG Ab, 2× weekly for 3 weeks (arrows indicateAb treatment). C) Tumor volumes are shown. *** indicate p-value<0.001(ANOVA) for aPD-L1+Rat IgG2b compared to αPD-L1+aPVRIG treated groups.Arrows indicate when antibodies were dosed. D. Survival analysis ofcomplete responder's mice. * indicate p value<0.05 (Log-rank test) forαPD-L1+Rat IgG2b compared to αPD-L1+αPVRIG treated groups. Onerepresentative study of 3 studies are shown. E. C57BL/6 or TIGIT^(−/−)mice were subcutaneously injected with 1×10⁵ B16/Db-hmgp100 cells. Micewere treated 2× weekly for 3 weeks with the designated mAb starting onthe day of inoculation (day 0). E. Tumor volumes were measured 2× weeklyand average±SEM is shown. Tumor growth inhibition as measured atindicated days compared to control WT+mIgG1 isotype control. ***indicate p-value<0.001 for TIGIT^(−/−)+aPVRIG compared to WT+mIgG1isotype control. Arrows indicate when antibodies were dosed. F.Individual tumor growth curves for each mouse is shown. Onerepresentative experiment out of 2 performed is shown.

FIG. 90A-90F. PVRIG is expressed on T and NK cells of TILS in humancancer. A) Expression of PVRIG, TIGIT, CD96, and PD-1 on CD4 T cellsubsets from healthy donor PBMCs is shown. Mean±SEM is shown. B) Human Tcells were co-cultured with allogeneic PBMCs and expression of PVRIGprotein on CD4 and CD8 T cells shown (top). C) Tumors were dissociatedand single cells were activated with anti-CD3 and anti-CD28. Expressionof PVRIG (blue) relative to IgG isotype control (red) was assessed onday 0 (directly ex vivo) and day 5 post activation. D) Expression ofPVRIG on NK cells from dissociated human tumors is shown. Each dotrepresents a distinct tumor from an individual patient. Mean±95%confidence internal is shown. D) Dissociated tumor cells were activatedwith anti-CD3 and anti-CD28 beads for 5 days. Expression of PVRIG (blue)relative to IgG control (red) on CD4 and CD8 T cells on day 0 directlyex vivo and on day 5 post activation is shown for 2 dissociated tumorsamples. E) Expression of PVRIG was assessed on CD4 and CD8 T cells fromdissociated tumors and from dissociated donor-matched normal adjacenttissue. Each line represents matched tissues obtained from an individualpatient. A paired student's t-test was performed. F) A correlationanalysis of the magnitude of PVRIG, TIGIT, and PD-1 fold expressionrelative to IgG isotype control on CD4 and CD8 T cells from tumors isshown. Each dot represents an individual tumor sample. A Spearman'scorrelation coefficient and p value are shown.

FIG. 91. Expression of PVRL2 is enhanced in colon, skin, and breastcancers. A) Photomicrographs showing the binding of Sigma anti humanPVRL2 antibody to FFPE sections of positive cells, CHO-S human PVRL2(right) compare to negative cells, CHO-S (left), following antigenretrieval at pH9. B) Anti-PVRL2 antibody was tested on a panel of PVRL2⁺(HT29, MCF7, PC3, PANC1, RT4, NCI-H1573) and PVRL2⁻ (Jurkat, OPM2,Daudi, CA46) cell lines. C-F) Example expression of PVRL2 in lung normaland cancer tissues. C) Normal tissue showing no staining. D) LungAdenocarcinoma showing partial positive staining. E) Lung adenocarcinomashowing positive staining. F) Lung adenocarcinoma showing strongpositive staining.

FIG. 92. PVRL2 is upregulated on TAMs and CD45⁻ cells in the tumor ascompared to normal adjacent tissue. Expression of PVRL2 on CD45⁻ cellsand TAMs from donor matched tumor and normal adjacent tissue is shown. Apaired student's t-test p value is shown.

FIGS. 93A and 93B. PVRIG and PVRL2 are co-expressed in the same tumorsample. PVRIG expression on CD4 T cells (A) and NK cells (B) is plottedagainst PVRL2 expression on TAMS for an individual tumor.

FIG. 94A-94D. Activity of CHA.7.518.1.H4(S241P) on human T cells. A)Expression of PVRIG on CD4 T cells activated with CHO cells expressingcell surface bound anti-CD3 and PVRL2. B) Expression of HLA-A2, B-2m,and PVRL2 are shown on CHO-S parental and engineered CHO-S cell lines.Fold expression relative to isotype is depicted by the number. C) CHOcells ectopically expressing cell surface bound anti-CD3 and PVRL2 wereco-cultured with purified CD8 T cells in the presence of varyingconcentrations of anti-PVRIG Ab or relevant IgG control. % Proliferationis shown. Each dot represents an average of triplicate values. D) CHOcells ectopically expression HLA-A2/B2m and PVRL2 were co-cultured with2 gp100 specific T cell lines (TIL F4, TIL 209) in the presence of 1ug/ml gp100 and varying concentrations of anti-PVRIG antibody orrelevant IgG control. TNF-α concentrations on day 3 of co-culture isdown. Each value represents an average of triplicates.

FIG. 95A-95J. Characterization of mPVRIG binding interactions and asurrogate anti-mPVRIG antibody. A, B) Binding of mPVRIG to mPVRL2 wasassessed by surface plasmon resonance. C) Soluble receptor Fc or controlproteins were incubated in a dose response with immobilized mPVRL2 HISin an ELISA format. Bound receptor Fc is shown. D) Soluble PVRL2 HISprotein was incubated in a dose response with PVRIG Fc or DNAM Fc coatedplates. E) Binding of mPVRIG Fc or control Fc fusion protein to B16-F10cell line transfected with mPVRL2 siRNA, mPVRsRNA, or scrambled siRNAtransfection is shown. F) Affinity characterization of rat anti-mousePVRIG mAb was performed by examining the binding of anti-mPVRIG toHEK293 cells overexpressing mPVRIG. G) Affinity characterization of ratanti-mouse PVRIG mAb was performed by examining the of anti-mPVRIG toD10.G4.1 cell line endogenously expressing mPVRIG vs isotype control ratIgG is shown. H) Binding of anti-mPVRIG to D10.G4.1 cells transfectedwith mouse PVRIG-siRNA (green histogram) vs scr siRNA (orangehistogram). I) Binding of mPVRIG Fc pre-incubated with anti-mPVRIG Ab toB16-F10 cells, which endogenously express PVRL2

FIG. 96. Generation of transgenic PVRIG and TIGIT knockout mice. ThePVRIG conditional knockout and Tigit knockout mouse lines were generatedby Ozgene Pty Ltd (Bentley W A, Australia). A) The targeting constructin which PVRIG exons 1 to 4 were floxed was electroporated into aC57BL/6 ES cell line, Bruce4 (Koentgen et al., Int Immunol 5: 957-964,1993). B) The targeting construct in which the coding region of Tigitexon 1 (including the ATG) and exons 2 and 3 were replaced with anFRT-flanked neo cassette was electroporated into a C57BL/6 ES cell line,Bruce4. Homologous recombinant ES cell clones were identified bySouthern hybridization and injected into goGermline blastocysts(Koentgen et al., genesis 54: 326-333, 2016). Male chimeric mice wereobtained and crossed to C57BL/6J females to establish heterozygousgermline offspring on C57BL/6 background. The germline mice were crossedto a ubiquitous FLP C57BL/6 mouse line to remove the FRT flankedselectable marker cassette and generate the conditional or knockoutalleles (for PVRIG and Tigit, respectively). For PVRIG knockout, micewere further crossed to a ubiquitous Cre C57BL/6 mouse line to removethe loxP flanked exons and generate the knockout allele.

FIG. 97A-97I. PVRIG knockout mice are immune-phenotypically similar towild-type mice. Mice (n=5 per wild-type and PVRIG knockout cohorts) wereeuthanized prior to venous blood being collected inanti-coagulant-coated tubes and harvesting of organs. Single cells wererecovered from freshly harvested bone marrow, thymus, spleen, cutaneousand mesenteric lymph nodes. Cells were stained withfluorochrome-conjugated surface marker antibodies and acquired on a BDLSR Fortessa flow cytometer. Panels illustrate comparable frequencies ofmyeloid cells (A), dendritic cells (B), B cells (C), T cells (D), CD4 Tcells (E), CD8 T cells (F), and NK cells (G) across lymphoid tissuetypes. (H-I) Whole venous blood was run on a Hemavet 950 veterinaryhematology system to compare differential counts and frequencies ofblood cell subsets from wild-type and PVRIG deficient mice.

FIG. 98A-98I. Increased T cell effector function in PVRIG^(−/−) micetreated with anti-PDL1 compared to WT with anti-PD-L1. MC38 tumors wereinoculated into WT or PVRIG^(−/−) mice and were subsequently treatedwith anti-PD-L1 or rat IgG2b isotype control. On day 18, CD45+ tumorinfiltrating lymphocytes were purified from tumors, RNA extracted, andtranscript profiling performed. Several T cell related genes are shown,with each dot representing an individual mouse. Student's t test pvalues are shown.

FIG. 99. Anti-TIGIT and anti-PVRIG antibodies induce tumor cell killing.An in vitro co-culture assay with human CMV-specific CD8+ T cellsexpanded was utilized to assess the effect of the benchmark anti-TIGITantibody and CHA.7.518.1.H4(S241P) on antigen-specific tumor cellkilling. HLA-A2+ target cell lines used in the assay were the Mel624 (A)and Panc05.04 (B). Synagis hIgG4 is the isotype control antibody.Luciferase activity in the target cells was measured with the Bio-Gloluciferase substrate. Representative data (n≥2) shows the percentspecific killing (mean+/−standard deviation) of Mel624 or Panc05.04cells after a 16-hour co-culture with human CMV-specific CD8+ T cellsfrom three different donors.

FIG. 100. Dose-dependent tumor cell killing of anti-TIGIT antibodieswith CHA.7.518.1.H4(S241P). An in vitro co-culture assay with humanCMV-specific CD8+ T cells was utilized to assess the effect of twodifferent anti-TIGIT antibodies, BM26 and CPA.9.086 when combined withCHA.7.518.1.H4(S241P) on antigen-specific Mel624 cell killing.Luciferase activity in the target cells was measured with the Bio-Gloluciferase substrate. Representative data (n≥2) shows the percentspecific killing (mean+/−standard deviation) of Mel624 cells after a16-hour co-culture with human CMV-specific CD8+ T cells from one donor.

FIG. 101. CPA.9.086 CDR sequences, IMGT and Kabat numbering.

FIG. 102. Anti-TIGIT hIgG4+CHA.7.518.1.H4(S241P) combination inducestumor cell killing. Co-culture of CMV-reactive CD8+ T cells with Mel624PVR, PVRL2 & luciferase OE Single dose of 10 μg/ml aTIGIT Ab and 10μg/ml CHA.7.518.1.H4(S241P) with CMV-reactive donor 4, while dosetitration starting at 0.5 μg/ml aTIGIT Ab and 10 μg/mlCHA.7.518.1.H4(S241P) with CMV-reactive donor 156.

V. DETAILED DESCRIPTION OF THE INVENTION

A. Overview

The present invention provides a number of useful antibodies, for usealone or in combination, for treatment of cancer. Cancer can beconsidered as an inability of the patient to recognize and eliminatecancerous cells. In many instances, these transformed (e.g. cancerous)cells counteract immunosurveillance. There are natural controlmechanisms that limit T-cell activation in the body to preventunrestrained T-cell activity, which can be exploited by cancerous cellsto evade or suppress the immune response. Restoring the capacity ofimmune effector cells-especially T cells—to recognize and eliminatecancer is the goal of immunotherapy. The field of immuno-oncology,sometimes referred to as “immunotherapy” is rapidly evolving, withseveral recent approvals of T cell checkpoint inhibitory antibodies suchas Yervoy, Keytruda and Opdivo. These antibodies are generally referredto as “checkpoint inhibitors” because they block normally negativeregulators of T cell immunity. It is generally understood that a varietyof immunomodulatory signals, both costimulatory and coinhibitory, can beused to orchestrate an optimal antigen-specific immune response.Generally, these antibodies bind to checkpoint inhibitor proteins suchas CTLA-4 or PD-1, which under normal circumstances prevent or suppressactivation of cytotoxic T cells (CTLs). By inhibiting the checkpointprotein, for example through the use of antibodies that bind theseproteins, an increased T cell response against tumors can be achieved.That is, these cancer checkpoint proteins suppress the immune response;when the proteins are blocked, for example using antibodies to thecheckpoint protein, the immune system is activated, leading to immunestimulation, resulting in treatment of conditions such as cancer andinfectious disease.

The present invention is directed to the use of antibodies to additionalcheckpoint proteins, PVRIG and TIGIT. PVRIG is expressed on the cellsurface of NK and T-cells and shares several similarities to other knownimmune checkpoints. The identification and methods used to show thatPVRIG is a checkpoint receptor are discussed in WO2016/134333, expresslyincorporated herein by reference. Antibodies to human PVRIG that blockthe interaction and/or binding of PVLR2 are provided herein. When PVRIGis bound by its ligand (PVRL2), an inhibitory signal is elicited whichacts to attenuate the immune response of NK and T-cells against a targetcell (i.e. analogous to PD-1/PDL1). Blocking the binding of PVRL2 toPVRIG shuts-off this inhibitory signal of PVRIG and as a resultmodulates the immune response of NK and T-cells. Utilizing an antibodyagainst PVRIG that blocks binding to PVRL2 is a therapeutic approachthat enhances the killing of cancer cells by NK and T-cells. Blockingantibodies have been generated which bind PVRIG and block the binding ofits ligand, PVRL2. Anti-PVRIG antibodies in combination with othercheckpoint inhibitor antibodies such as PD-1 are provided.

Similarly, TIGIT has been shown to also have attributes of a checkpointreceptor, and the present invention provides anti-TIGIT antibodies thatblock the interaction and/or binding of TIGIT to PVR are provided. WhenTIGIT is bound by its ligand (PVR), an inhibitory signal is elicitedwhich acts to attenuate the immune response of NK and T-cells against atarget cell (i.e. analogous to PD-1/PDL1). Blocking the binding of PVRto TIGIT shuts-off this inhibitory signal of TIGIT and as a resultmodulates the immune response of NK and T-cells. Utilizing an antibodyagainst TIGIT that blocks binding to PVR is a therapeutic approach thatenhances the killing of cancer cells by NK and T-cells. Blockingantibodies have been generated which bind TIGIT and block the binding ofits ligand, PVR. Anti-TIGIT antibodies in combination with othercheckpoint inhibitor antibodies such as PD-1 are provided.

Additionally, the invention provides combinations of anti-PVRIG andanti-TIGIT antibodies for use in the treatment of cancer.

B. Definitions

In order that the application may be more completely understood, severaldefinitions are set forth below. Such definitions are meant to encompassgrammatical equivalents.

By “ablation” herein is meant a decrease or removal of activity. In someembodiments, it is useful to remove activity from the constant domainsof the antibodies. Thus, for example, “ablating FcγR binding” means theFc region amino acid variant has less than 50% starting binding ascompared to an Fc region not containing the specific variant, with lessthan 70-80-90-95-98% loss of activity being preferred, and in general,with the activity being below the level of detectable binding in aBiacore assay. As shown in FIG. 50, one ablation variant in the IgG1constant region is the N297A variant, which removes the nativeglycosylation site and significantly reduces the FcγRIIIa binding andthus reduces the antibody dependent cell-mediated cytotoxicity (ADCC).

By “antigen binding domain” or “ABD” herein is meant a set of sixComplementary Determining Regions (CDRs) that, when present as part of apolypeptide sequence, specifically binds a target antigen as discussedherein. Thus, a “TIGIT antigen binding domain” binds TIGIT antigen (thesequence of which is shown in FIG. 51) as outlined herein. Similarly, a“PVRIG antibody binding domain” binds PVRIG antigen (the sequence ofwhich is shown in FIG. 1) as outlined herein. As is known in the art,these CDRs are generally present as a first set of variable heavy CDRs(vhCDRs or V_(H)CDRs) and a second set of variable light CDRs (vlCDRs orV_(L)CDR5), each comprising three CDRs: vhCDR1, vhCDR2, vhCDR3 for theheavy chain and vlCDR1, vlCDR2 and vlCDR3 for the light. The CDRs arepresent in the variable heavy and variable light domains, respectively,and together form an Fv region. Thus, in some cases, the six CDRs of theantigen binding domain are contributed by a variable heavy and variablelight chain. In a “Fab” format, the set of 6 CDRs are contributed by twodifferent polypeptide sequences, the variable heavy domain (vh or V_(H);containing the vhCDR1, vhCDR2 and vhCDR3) and the variable light domain(vl or V_(L); containing the vlCDR1, vlCDR2 and vlCDR3), with theC-terminus of the vh domain being attached to the N-terminus of the CH1domain of the heavy chain and the C-terminus of the vl domain beingattached to the N-terminus of the constant light domain (and thusforming the light chain).

By “modification” herein is meant an amino acid substitution, insertion,and/or deletion in a polypeptide sequence or an alteration to a moietychemically linked to a protein. For example, a modification may be analtered carbohydrate or PEG structure attached to a protein. By “aminoacid modification” herein is meant an amino acid substitution,insertion, and/or deletion in a polypeptide sequence. For clarity,unless otherwise noted, the amino acid modification is always to anamino acid coded for by DNA, e.g. the 20 amino acids that have codons inDNA and RNA.

By “amino acid substitution” or “substitution” herein is meant thereplacement of an amino acid at a particular position in a parentpolypeptide sequence with a different amino acid. In particular, in someembodiments, the substitution is to an amino acid that is not naturallyoccurring at the particular position, either not naturally occurringwithin the organism or in any organism. For example, the substitutionN297A refers to a variant polypeptide, in this case an Fc variant, inwhich the asparagine at position 297 is replaced with alanine. Forclarity, a protein which has been engineered to change the nucleic acidcoding sequence but not change the starting amino acid (for exampleexchanging CGG (encoding arginine) to CGA (still encoding arginine) toincrease host organism expression levels) is not an “amino acidsubstitution”; that is, despite the creation of a new gene encoding thesame protein, if the protein has the same amino acid at the particularposition that it started with, it is not an amino acid substitution.

By “amino acid insertion” or “insertion” as used herein is meant theaddition of an amino acid sequence at a particular position in a parentpolypeptide sequence. For example, −233E or 233E designates an insertionof glutamic acid after position 233 and before position 234.Additionally, −233ADE or A233ADE designates an insertion of AlaAspGluafter position 233 and before position 234.

By “amino acid deletion” or “deletion” as used herein is meant theremoval of an amino acid sequence at a particular position in a parentpolypeptide sequence. For example, E233− or E233#, E233( ) or E233deldesignates a deletion of glutamic acid at position 233. Additionally,EDA233− or EDA233# designates a deletion of the sequence GluAspAla thatbegins at position 233.

By “variant protein” or “protein variant”, or “variant” as used hereinis meant a protein that differs from that of a parent protein by virtueof at least one amino acid modification. Protein variant may refer tothe protein itself, a composition comprising the protein, or the aminosequence that encodes it. Preferably, the protein variant has at leastone amino acid modification compared to the parent protein, e.g. fromabout one to about seventy amino acid modifications, and preferably fromabout one to about five amino acid modifications compared to the parent.As described below, in some embodiments the parent polypeptide, forexample an Fc parent polypeptide, is a human wild type sequence, such asthe Fc region from IgG1, IgG2, IgG3 or IgG4, although human sequenceswith variants can also serve as “parent polypeptides”. The proteinvariant sequence herein will preferably possess at least about 80%identity with a parent protein sequence, and most preferably at leastabout 90% identity, more preferably at least about 95-98-99% identity.Variant protein can refer to the variant protein itself, compositionscomprising the protein variant, or the DNA sequence that encodes it.Accordingly, by “antibody variant” or “variant antibody” as used hereinis meant an antibody that differs from a parent antibody by virtue of atleast one amino acid modification, “IgG variant” or “variant IgG” asused herein is meant an antibody that differs from a parent IgG (again,in many cases, from a human IgG sequence) by virtue of at least oneamino acid modification, and “immunoglobulin variant” or “variantimmunoglobulin” as used herein is meant an immunoglobulin sequence thatdiffers from that of a parent immunoglobulin sequence by virtue of atleast one amino acid modification. “Fc variant” or “variant Fc” as usedherein is meant a protein comprising an amino acid modification in an Fcdomain. The Fc variants of the present invention are defined accordingto the amino acid modifications that compose them. Thus, for example,S241P or S228P is a hinge variant with the substitution proline atposition 228 relative to the parent IgG4 hinge polypeptide, wherein thenumbering S228P is according to the EU index and the S241P is the Kabatnumbering. The EU index or EU index as in Kabat or EU numbering schemerefers to the numbering of the EU antibody (Edelman et al., 1969, ProcNatl Acad Sci USA 63:78-85, hereby entirely incorporated by reference.)The modification can be an addition, deletion, or substitution.Substitutions can include naturally occurring amino acids and, in somecases, synthetic amino acids. Examples include U.S. Pat. No. 6,586,207;WO 98/48032; WO 03/073238; US2004-0214988A1; WO 05/35727A2; WO05/74524A2; J. W. Chin et al., (2002), Journal of the American ChemicalSociety 124:9026-9027; J. W. Chin, & P. G. Schultz, (2002), ChemBioChem11:1135-1137; J. W. Chin, et al., (2002), PICAS United States of America99:11020-11024; and, L. Wang, & P. G. Schultz, (2002), Chem. 1-10, allentirely incorporated by reference.

As used herein, “protein” herein is meant at least two covalentlyattached amino acids, which includes proteins, polypeptides,oligopeptides and peptides. The peptidyl group may comprise naturallyoccurring amino acids and peptide bonds, or synthetic peptidomimeticstructures, i.e. “analogs”, such as peptoids (see Simon et al., PNAS USA89(20):9367 (1992), entirely incorporated by reference). The amino acidsmay either be naturally occurring or synthetic (e.g. not an amino acidthat is coded for by DNA); as will be appreciated by those in the art.For example, homo-phenylalanine, citrulline, ornithine and noreleucineare considered synthetic amino acids for the purposes of the invention,and both D- and L- (R or S) configured amino acids may be utilized. Thevariants of the present invention may comprise modifications thatinclude the use of synthetic amino acids incorporated using, forexample, the technologies developed by Schultz and colleagues, includingbut not limited to methods described by Cropp & Shultz, 2004, TrendsGenet. 20(12):625-30, Anderson et al., 2004, Proc Natl Acad Sci USA 101(2):7566-71, Zhang et al., 2003, 303(5656):371-3, and Chin et al., 2003,Science 301(5635):964-7, all entirely incorporated by reference. Inaddition, polypeptides may include synthetic derivatization of one ormore side chains or termini, glycosylation, PEGylation, circularpermutation, cyclization, linkers to other molecules, fusion to proteinsor protein domains, and addition of peptide tags or labels.

By “residue” as used herein is meant a position in a protein and itsassociated amino acid identity. For example, Asparagine 297 (alsoreferred to as Asn297 or N297) is a residue at position 297 in the humanantibody IgG1.

By “Fab” or “Fab region” as used herein is meant the polypeptide thatcomprises the VH, CH1, VL, and CL immunoglobulin domains. Fab may referto this region in isolation, or this region in the context of a fulllength antibody or antibody fragment.

By “Fv” or “Fv fragment” or “Fv region” as used herein is meant apolypeptide that comprises the VL and VH domains of a single antibody.As will be appreciated by those in the art, these generally are made upof two chains.

By “single chain Fv” or “scFv” herein is meant a variable heavy domaincovalently attached to a variable light domain, generally using a scFvlinker as discussed herein, to form a scFv or scFv domain. A scFv domaincan be in either orientation from N- to C-terminus (vh-linker-vl orvl-linker-vh). In general, the linker is a scFv linker as is generallyknown in the art, with the linker peptide predominantly including thefollowing amino acid residues: Gly, Ser, Ala, or Thr. The linker peptideshould have a length that is adequate to link two molecules in such away that they assume the correct conformation relative to one another sothat they retain the desired activity. In one embodiment, the linker isfrom about 1 to 50 amino acids in length, preferably about 1 to 30 aminoacids in length. In one embodiment, linkers of 1 to 20 amino acids inlength may be used, with from about 5 to about 10 amino acids findinguse in some embodiments. Useful linkers include glycine-serine polymers,including for example (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, where n isan integer of at least one (and generally from 3 to 4), glycine-alaninepolymers, alanine-serine polymers, and other flexible linkers.Alternatively, a variety of nonproteinaceous polymers, including but notlimited to polyethylene glycol (PEG), polypropylene glycol,polyoxyalkylenes, or copolymers of polyethylene glycol and polypropyleneglycol, may find use as linkers, that is may find use as linkers.

By “IgG subclass modification” or “isotype modification” as used hereinis meant an amino acid modification that converts one amino acid of oneIgG isotype to the corresponding amino acid in a different, aligned IgGisotype. For example, because IgG1 comprises a tyrosine and IgG2 aphenylalanine at EU position 296, a F296Y substitution in IgG2 isconsidered an IgG subclass modification. Similarly, because IgG1 has aproline at position 241 and IgG4 has a serine there, an IgG4 moleculewith a S241P is considered an IgG subclass modification. Note thatsubclass modifications are considered amino acid substitutions herein.

By “non-naturally occurring modification” as used herein is meant anamino acid modification that is not isotypic. For example, because noneof the IgGs comprise AN asparagine at position 297, the substitutionN297A in IgG1, IgG2, IgG3, or IgG4 (or hybrids thereof) is considered anon-naturally occurring modification.

By “amino acid” and “amino acid identity” as used herein is meant one ofthe 20 naturally occurring amino acids that are coded for by DNA andRNA.

By “effector function” as used herein is meant a biochemical event thatresults from the interaction of an antibody Fc region with an Fcreceptor or ligand. Effector functions include but are not limited toADCC, ADCP, and CDC.

By “IgG Fc ligand” as used herein is meant a molecule, preferably apolypeptide, from any organism that binds to the Fc region of an IgGantibody to form an Fc/Fc ligand complex. Fc ligands include but are notlimited to FcγRIs, FcγRIIs, FcγRIIIs, FcRn, C1q, C3, mannan bindinglectin, mannose receptor, staphylococcal protein A, streptococcalprotein G, and viral FcγR. Fc ligands also include Fc receptor homologs(FcRH), which are a family of Fc receptors that are homologous to theFcγRs (Davis et al., 2002, Immunological Reviews 190:123-136, entirelyincorporated by reference). Fc ligands may include undiscoveredmolecules that bind Fc. Particular IgG Fc ligands are FcRn and Fc gammareceptors. By “Fc ligand” as used herein is meant a molecule, preferablya polypeptide, from any organism that binds to the Fc region of anantibody to form an Fc/Fc ligand complex.

By “parent polypeptide” as used herein is meant a starting polypeptidethat is subsequently modified to generate a variant. The parentpolypeptide may be a naturally occurring polypeptide, or a variant orengineered version of a naturally occurring polypeptide. Parentpolypeptide may refer to the polypeptide itself, compositions thatcomprise the parent polypeptide, or the amino acid sequence that encodesit. Accordingly, by “parent immunoglobulin” as used herein is meant anunmodified immunoglobulin polypeptide that is modified to generate avariant, and by “parent antibody” as used herein is meant an unmodifiedantibody that is modified to generate a variant antibody. It should benoted that “parent antibody” includes known commercial, recombinantlyproduced antibodies as outlined below.

By “Fc” or “Fc region” or “Fc domain” as used herein is meant thepolypeptide comprising the constant region of an antibody excluding thefirst constant region immunoglobulin domain and in some cases, part ofthe hinge. Thus Fc refers to the last two constant region immunoglobulindomains of IgA, IgD, and IgG, the last three constant regionimmunoglobulin domains of IgE and IgM, and the flexible hinge N-terminalto these domains. For IgA and IgM, Fc may include the J chain. For IgG,the Fc domain comprises immunoglobulin domains Cγ2 and Cγ3 (Cγ2 and Cγ3)and the lower hinge region between Cγ1 (Cγ1) and Cγ2 (Cγ2). Although theboundaries of the Fc region may vary, the human IgG heavy chain Fcregion is usually defined to include residues C226 or P230 to itscarboxyl-terminus, wherein the numbering is according to the EU index asin Kabat. In some embodiments, as is more fully described below, aminoacid modifications are made to the Fc region, for example to alterbinding to one or more FcγR receptors or to the FcRn receptor.

By “heavy constant region” herein is meant the CH1-hinge-CH2-CH3 portionof an antibody.

By “position” as used herein is meant a location in the sequence of aprotein. Positions may be numbered sequentially, or according to anestablished format, for example the EU index for antibody numbering.

By “target antigen” as used herein is meant the molecule that is boundspecifically by the variable region of a given antibody. In the presentcase, one target antigen of interest herein is TIGIT, usually humanTIGIT and optionally cyno TIGIT, as defined below. Another targetantigen of interest is PVRIG, usually human PVRIG and optionally cynoPVRIG, as defined below.

By “target cell” as used herein is meant a cell that expresses a targetantigen.

By “variable region” as used herein is meant the region of animmunoglobulin that comprises one or more Ig domains substantiallyencoded by any of the Vκ (V.kappa), Vλ (V.lamda), and/or VH genes thatmake up the kappa, lambda, and heavy chain immunoglobulin genetic locirespectively.

By “wild type or WT” herein is meant an amino acid sequence or anucleotide sequence that is found in nature, including allelicvariations. A WT protein has an amino acid sequence or a nucleotidesequence that has not been intentionally modified.

The antibodies of the present invention are generally isolated orrecombinant. “Isolated,” when used to describe the various polypeptidesdisclosed herein, means a polypeptide that has been identified andseparated and/or recovered from a cell or cell culture from which it wasexpressed. Ordinarily, an isolated polypeptide will be prepared by atleast one purification step. An “isolated antibody,” refers to anantibody which is substantially free of other antibodies havingdifferent antigenic specificities. “Recombinant” means the antibodiesare generated using recombinant nucleic acid techniques in exogeneoushost cells.

“Specific binding” or “specifically binds to” or is “specific for” aparticular antigen or an epitope means binding that is measurablydifferent from a non-specific interaction. Specific binding can bemeasured, for example, by determining binding of a molecule compared tobinding of a control molecule, which generally is a molecule of similarstructure that does not have binding activity. For example, specificbinding can be determined by competition with a control molecule that issimilar to the target.

Specific binding for a particular antigen or an epitope can beexhibited, for example, by an antibody having a KD for an antigen orepitope of at least about 10⁻⁹ M, at least about 10⁻¹⁰ M, at least about10⁻¹¹ M, at least about 10⁻¹² M, at least about 10⁻¹³ M, at least about10⁻¹⁴ M, at least about 10⁻¹⁵ M, where KD refers to a dissociation rateof a particular antibody-antigen interaction. Typically, an antibodythat specifically binds an antigen will have a KD that is 20-, 50-,100-, 500-, 1000-, 5,000-, 10,000- or more times greater for a controlmolecule relative to the antigen or epitope.

Also, specific binding for a particular antigen or an epitope can beexhibited, for example, by an antibody having a KA or Ka for an antigenor epitope of at least 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- ormore times greater for the epitope relative to a control, where KA or Karefers to an association rate of a particular antibody-antigeninteraction. Binding affinity is generally measured using surfaceplasmon resonance (e.g. Biacore assay) and flow cytometry withantigen-expressing cells.

C. Sequences

The sequence listing provides a number of sequences based on the Formatof FIG. 53; reference is made to FIG. 4 of U.S. Ser. No. 62/513,916(hereby expressly incorporated by reference) as a guide to the labelingof the sequences. The variable heavy domain is labeled with theidentifier (e.g. “CPA.0.86”), with the next sequence following theformat of FIG. 53 of the present specification (identical to the formatof FIG. 4, referenced above), in that the next sequence identifier is tothe vhCDR1, the next to vhCDR2, with vhCDR3, the full length heavychain, the variable light domain, vlCDR1, vlCDR2, vlCDR3 and the fulllength light chain. Thus an individual antibody has 10 associatedsequence identifiers.). Included in the sequence listing are thesequences of BM26 mouse IgG1 (BM26-M1) (WO2016/028656A1, Clone 3106) andBM29 mouse IgG1 (BM29-M1) (US2016/0176963A1, Clone 22G2). Unless noted,the full length HC sequences of the TIGIT antibodies are in theH4(S241P) format.

D. PVRIG Proteins

The present invention provides antibodies that specifically bind toPVRIG proteins and prevent activation by its ligand protein, PVRL2, ahuman plasma membrane glycoprotein. PVRIG, also called PoliovirusReceptor Related Immunoglobulin Domain Containing Protein, Q6DKI7 orC7orf15, relates to amino acid and nucleic acid sequences shown inRefSeq accession identifier NP_076975, shown in FIG. 1. The sequence ofhuman Poliovirus receptor-related 2 protein (PVLR2, also known asnectin-2, CD112 or herpesvirus entry mediator B, (HVEB)), the bindingpartner of PVRIG (as shown in Example 5 of US Publication 2016/0244521),is shown in FIG. 2. The antibodies of the invention are specific for thePVRIG extracellular domain such that the binding of PVRIG and PVLR2 isblocked.

PVRIG is a transmembrane domain protein of 326 amino acids in length,with a signal peptide (spanning from amino acid 1 to 40), anextracellular domain (spanning from amino acid 41 to 171), atransmembrane domain (spanning from amino acid 172 to 190) and acytoplasmic domain (spanning from amino acid 191 to 326). There are twomethionines that can be start codons, but the mature proteins areidentical.

Accordingly, as used herein, the term “PVRIG” or “PVRIG protein” or“PVRIG polypeptide” may optionally include any such protein, orvariants, conjugates, or fragments thereof, including but not limited toknown or wild type PVRIG, as described herein, as well as any naturallyoccurring splice variants, amino acid variants or isoforms, and inparticular the ECD fragment of PVRIG.

As noted herein and more fully described below, anti-PVRIG antibodies(including antigen-binding fragments) that both bind to PVRIG andprevent activation by PVRL2 (e.g. most commonly by blocking theinteraction of PVRIG and PVLR2), are used to enhance T cell and/or NKcell activation and be used in treating diseases such as cancer andpathogen infection.

E. TIGIT Proteins

The present invention provides antibodies that specifically bind toTIGIT proteins and prevent activation by its ligand protein, PVR,poliovirus receptor (aka CD155) a human plasma membrane glycoprotein.TIGIT, or T cell immunoreceptor with Ig and ITIM domains, is aco-inhibiotry receptor protein also known as WUCAM, Vstm3 or Vsig9.TIGIT has an immunoglobulin variable domain, a transmembrane domain, andan immunoreceptor tyrosine-based inhibitory motif (ITIM) and containssignature sequence elements of the PVR protein family. The extracellulardomain (ECD) sequences of TIGIT and of PVR are shown in FIG. 51. Theantibodies of the invention are specific for the TIGIT ECD such that thebinding of TIGIT and PVR is blocked

Accordingly, as used herein, the term “TIGIT” or “TIGIT protein” or“TIGIT polypeptide” may optionally include any such protein, orvariants, conjugates, or fragments thereof, including but not limited toknown or wild type TIGIT, as described herein, as well as any naturallyoccurring splice variants, amino acid variants or isoforms, and inparticular the ECD fragment of TIGIT.

As noted herein and more fully described below, anti-TIGIT antibodies(including antigen-binding fragments) that both bind to TIGIT andprevent activation by PVR (e.g. most commonly by blocking theinteraction of TIGIT and PVR), are used to enhance T cell and/or NK cellactivation and be used in treating diseases such as cancer and pathogeninfection.

VI. ANTIBODIES

As is discussed below, the term “antibody” is used generally.Traditional antibody structural units typically comprise a tetramer.Each tetramer is typically composed of two identical pairs ofpolypeptide chains, each pair having one “light” (typically having amolecular weight of about 25 kDa) and one “heavy” chain (typicallyhaving a molecular weight of about 50-70 kDa). Human light chains areclassified as kappa and lambda light chains. The present invention isdirected to monoclonal antibodies that generally are based on the IgGclass, which has several subclasses, including, but not limited to IgG1,IgG2, IgG3, and IgG4. In general, IgG1, IgG2 and IgG4 are used morefrequently than IgG3. It should be noted that IgG1 has differentallotypes with polymorphisms at 356 (D or E) and 358 (L or M). Thesequences depicted herein use the 356D/358M allotype, however the otherallotype is included herein. That is, any sequence inclusive of an IgG1Fc domain included herein can have 356E/358L replacing the 356D/358Mallotype.

The amino-terminal portion of each chain includes a variable region ofabout 100 to 110 or more amino acids primarily responsible for antigenrecognition, generally referred to in the art and herein as the “Fvdomain” or “Fv region”. In the variable region, three loops are gatheredfor each of the V domains of the heavy chain and light chain to form anantigen-binding site. Each of the loops is referred to as acomplementarity-determining region (hereinafter referred to as a “CDR”),in which the variation in the amino acid sequence is most significant.“Variable” refers to the fact that certain segments of the variableregion differ extensively in sequence among antibodies. Variabilitywithin the variable region is not evenly distributed. Instead, the Vregions consist of relatively invariant stretches called frameworkregions (FRs) of 15-30 amino acids separated by shorter regions ofextreme variability called “hypervariable regions” that are each 9-15amino acids long or longer.

Each VH and VL is composed of three hypervariable regions(“complementary determining regions,” “CDRs”) and four FRs, arrangedfrom amino-terminus to carboxy-terminus in the following order:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

The hypervariable region generally encompasses amino acid residues fromabout amino acid residues 24-34 (LCDR1; “L” denotes light chain), 50-56(LCDR2) and 89-97 (LCDR3) in the light chain variable region and aroundabout 31-35B (HCDR1; “H” denotes heavy chain), 50-65 (HCDR2), and 95-102(HCDR3) in the heavy chain variable region; Kabat et al., SEQUENCES OFPROTEINS OF IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991) and/or thoseresidues forming a hypervariable loop (e.g. residues 26-32 (LCDR1),50-52 (LCDR2) and 91-96 (LCDR3) in the light chain variable region and26-32 (HCDR1), 53-55 (HCDR2) and 96-101 (HCDR3) in the heavy chainvariable region; Chothia and Lesk (1987) J. Mol. Biol. 196:901-917.Specific CDRs of the invention are described below.

As will be appreciated by those in the art, the exact numbering andplacement of the CDRs can be different among different numberingsystems. However, it should be understood that the disclosure of avariable heavy and/or variable light sequence includes the disclosure ofthe associated (inherent) CDRs. Accordingly, the disclosure of eachvariable heavy region is a disclosure of the vhCDRs (e.g. vhCDR1, vhCDR2and vhCDR3) and the disclosure of each variable light region is adisclosure of the vlCDRs (e.g. vlCDR1, vlCDR2 and vlCDR3). A usefulcomparison of CDR numbering is as below, see Lafranc et al., Dev. Comp.Immunol. 27(1):55-77 (2003):

Kabat + Clothia IMGT Kabat AbM Chothia Contact vhCDR1 26-35 27-38 31-3526-35 26-32 30-35 vhCDR2 50-65 56-65 50-65 50-58 53-55 47-58 vhCDR3 95-102 105-117  95-102  95-102  96-101  93-101 vlCDR1 24-34 27-38 24-3424-34 26-32 30-36 vlCDR2 50-56 56-65 50-56 50-56 50-52 46-55 vlCDR389-97 105-117 89-97 89-97 91-96 89-96

Throughout the present specification, the Kabat numbering system isgenerally used when referring to a residue in the variable domain(approximately, residues 1-107 of the light chain variable region andresidues 1-113 of the heavy chain variable region) and the hinge and theEU numbering system for Fc regions (e.g, Kabat et al., supra (1991)).

The present invention provides a large number of different CDR sets. Inthis case, a “full CDR set” comprises the three variable light and threevariable heavy CDRs, e.g. a vlCDR1, vlCDR2, vlCDR3, vhCDR1, vhCDR2 andvhCDR3. These can be part of a larger variable light or variable heavydomain, respectfully. In addition, as more fully outlined herein, thevariable heavy and variable light domains can be on separate polypeptidechains, when a heavy and light chain is used, or on a single polypeptidechain in the case of scFv sequences.

The CDRs contribute to the formation of the antigen-binding, or morespecifically, epitope binding site of antibodies. “Epitope” refers to adeterminant that interacts with a specific antigen binding site in thevariable region of an antibody molecule known as a paratope. Epitopesare groupings of molecules such as amino acids or sugar side chains andusually have specific structural characteristics, as well as specificcharge characteristics. A single antigen may have more than one epitope.

The epitope may comprise amino acid residues directly involved in thebinding (also called immunodominant component of the epitope) and otheramino acid residues, which are not directly involved in the binding,such as amino acid residues which are effectively blocked by thespecifically antigen binding peptide; in other words, the amino acidresidue is within the footprint of the specifically antigen bindingpeptide.

Epitopes may be either conformational or linear. A conformationalepitope is produced by spatially juxtaposed amino acids from differentsegments of the linear polypeptide chain. A linear epitope is oneproduced by adjacent amino acid residues in a polypeptide chain.Conformational and non-conformational epitopes may be distinguished inthat the binding to the former but not the latter is lost in thepresence of denaturing solvents.

An epitope typically includes at least 3, and more usually, at least 5or 8-10 amino acids in a unique spatial conformation. Antibodies thatrecognize the same epitope can be verified in a simple immunoassayshowing the ability of one antibody to block the binding of anotherantibody to a target antigen, for example “binning.” As outlined below,the invention not only includes the enumerated antigen binding domainsand antibodies herein, but those that compete for binding with theepitopes bound by the enumerated antigen binding domains.

The carboxy-terminal portion of each chain defines a constant regionprimarily responsible for effector function. Kabat et al. collectednumerous primary sequences of the variable regions of heavy chains andlight chains. Based on the degree of conservation of the sequences, theyclassified individual primary sequences into the CDR and the frameworkand made a list thereof (see SEQUENCES OF IMMUNOLOGICAL INTEREST, 5thedition, NIH publication, No. 91-3242, E. A. Kabat et al., entirelyincorporated by reference).

In the IgG subclass of immunoglobulins, there are several immunoglobulindomains in the heavy chain. By “immunoglobulin (Ig) domain” herein ismeant a region of an immunoglobulin having a distinct tertiarystructure. Of interest in the present invention are the heavy chaindomains, including, the constant heavy (CH) domains and the hingedomains. In the context of IgG antibodies, the IgG isotypes each havethree CH regions. Accordingly, “CH” domains in the context of IgG are asfollows: “CH1” refers to positions 118-220 according to the EU index asin Kabat. “CH2” refers to positions 237-340 according to the EU index asin Kabat, and “CH3” refers to positions 341-447 according to the EUindex as in Kabat.

Another type of Ig domain of the heavy chain is the hinge region. By“hinge” or “hinge region” or “antibody hinge region” or “immunoglobulinhinge region” herein is meant the flexible polypeptide comprising theamino acids between the first and second constant domains of anantibody. Structurally, the IgG CH1 domain ends at EU position 220, andthe IgG CH2 domain begins at residue EU position 237. Thus for IgG theantibody hinge is herein defined to include positions 221 (D221 in IgG1)to 236 (G236 in IgG1), wherein the numbering is according to the EUindex as in Kabat.

The light chain generally comprises two domains, the variable lightdomain (containing the light chain CDRs and together with the variableheavy domains forming the Fv region), and a constant light chain region(often referred to as CL or Cκ). In general, either the constant lambdaor constant kappa domain can be used, with lambda generally finding usein the invention.

Another region of interest for additional substitutions, outlined below,is the Fc region.

A. Chimeric and Humanized Antibodies

In some embodiments, the antibodies herein can be derived from a mixturefrom different species, e.g. a chimeric antibody and/or a humanizedantibody. In general, both “chimeric antibodies” and “humanizedantibodies” refer to antibodies that combine regions from more than onespecies. For example, “chimeric antibodies” traditionally comprisevariable region(s) from a mouse (or rat, in some cases) and the constantregion(s) from a human. “Humanized antibodies” generally refer tonon-human antibodies that have had the variable-domain framework regionsswapped for sequences found in human antibodies. Generally, in ahumanized antibody, the entire antibody, except the CDRs, is encoded bya polynucleotide of human origin or is identical to such an antibodyexcept within its CDRs. The CDRs, some or all of which are encoded bynucleic acids originating in a non-human organism, are grafted into thebeta-sheet framework of a human antibody variable region to create anantibody, the specificity of which is determined by the engrafted CDRs.The creation of such antibodies is described in, e.g., WO 92/11018,Jones, 1986, Nature 321:522-525, Verhoeyen et al., 1988, Science239:1534-1536, all entirely incorporated by reference. “Backmutation” ofselected acceptor framework residues to the corresponding donor residuesis often required to regain affinity that is lost in the initial graftedconstruct (U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762;6,180,370; 5,859,205; 5,821,337; 6,054,297; 6,407,213, all entirelyincorporated by reference). The humanized antibody optimally also willcomprise at least a portion, and usually all, of an immunoglobulinconstant region, typically that of a human immunoglobulin, and thus willtypically comprise a human Fc region. Humanized antibodies can also begenerated using mice with a genetically engineered immune system. Roqueet al., 2004, Biotechnol. Prog. 20:639-654, entirely incorporated byreference. A variety of techniques and methods for humanizing andreshaping non-human antibodies are well known in the art (See Tsurushita& Vasquez, 2004, Humanization of Monoclonal Antibodies, MolecularBiology of B Cells, 533-545, Elsevier Science (USA), and referencescited therein, all entirely incorporated by reference). Humanizationmethods include but are not limited to methods described in Jones etal., 1986, Nature 321:522-525; Riechmann et al., 1988; Nature332:323-329; Verhoeyen et al., 1988, Science, 239:1534-1536; Queen etal., 1989, Proc Natl Acad Sci, USA 86:10029-33; He et al., 1998, J.Immunol. 160: 1029-1035; Carter et al., 1992, Proc Natl Acad Sci USA89:4285-9, Presta et al., 1997, Cancer Res. 57(20):4593-9; Gorman etal., 1991, Proc. Natl. Acad. Sci. USA 88:4181-4185; O'Connor et al.,1998, Protein Eng 11:321-8, all entirely incorporated by reference.Humanization or other methods of reducing the immunogenicity of nonhumanantibody variable regions may include resurfacing methods, as describedfor example in Roguska et al., 1994, Proc. Natl. Acad. Sci. USA91:969-973, entirely incorporated by reference.

Thus, the vhCDRs and vlCDRs from any of the enumerated antibodies hereinmay be humanized (or “rehumanized”, for those that were alreadyhumanized).

In certain embodiments, the antibodies of the invention comprise a heavychain variable region from a particular germline heavy chainimmunoglobulin gene and/or a light chain variable region from aparticular germline light chain immunoglobulin gene. For example, suchantibodies may comprise or consist of a human antibody comprising heavyor light chain variable regions that are “the product of” or “derivedfrom” a particular germline sequence. A human antibody that is “theproduct of” or “derived from” a human germline immunoglobulin sequencecan be identified as such by comparing the amino acid sequence of thehuman antibody to the amino acid sequences of human germlineimmunoglobulins and selecting the human germline immunoglobulin sequencethat is closest in sequence (i.e., greatest % identity) to the sequenceof the human antibody. A human antibody that is “the product of” or“derived from” a particular human germline immunoglobulin sequence maycontain amino acid differences as compared to the germline sequence, dueto, for example, naturally-occurring somatic mutations or intentionalintroduction of site-directed mutation. However, a humanized antibodytypically is at least 90% identical in amino acids sequence to an aminoacid sequence encoded by a human germline immunoglobulin gene andcontains amino acid residues that identify the antibody as being derivedfrom human sequences when compared to the germline immunoglobulin aminoacid sequences of other species (e.g., murine germline sequences). Incertain cases, a humanized antibody may be at least 95, 96, 97, 98 or99%, or even at least 96%, 97%, 98%, or 99% identical in amino acidsequence to the amino acid sequence encoded by the germlineimmunoglobulin gene excluding the CDRs. That is, the CDRs may be murine,but the framework regions of the variable region (either heavy or light)can be at least 96%, 97%, 98%, or 99% identical in amino acid sequenceto the framework amino acids encoded by one human germlineimmunoglobulin gene.

Typically, a humanized antibody derived from a particular human germlinesequence will display no more than 10-20 amino acid differences from theamino acid sequence encoded by the human germline immunoglobulin gene.In certain cases, the humanized antibody may display no more than 5, oreven no more than 4, 3, 2, or 1 amino acid difference from the aminoacid sequence encoded by the germline immunoglobulin gene (again, priorto the introduction of any variants herein; that is, the number ofvariants is generally low).

In one embodiment, the parent antibody has been affinity matured, as isknown in the art. Structure-based methods may be employed forhumanization and affinity maturation, for example as described in U.S.Ser. No. 11/004,590. Selection based methods may be employed to humanizeand/or affinity mature antibody variable regions, including but notlimited to methods described in Wu et al., 1999, J. Mol. Biol.294:151-162; Baca et al., 1997, J. Biol. Chem. 272(16):10678-10684;Rosok et al., 1996, J. Biol. Chem. 271(37): 22611-22618; Rader et al.,1998, Proc. Natl. Acad. Sci. USA 95: 8910-8915; Krauss et al., 2003,Protein Engineering 16(10):753-759, all entirely incorporated byreference. Other humanization methods may involve the grafting of onlyparts of the CDRs, including but not limited to methods described inU.S. Ser. No. 09/810,510; Tan et al., 2002, J. Immunol. 169:1119-1125;De Pascalis et al., 2002, J. Immunol. 169:3076-3084, all entirelyincorporated by reference.

B. Optional Antibody Engineering

The antibodies of the invention can be modified, or engineered, to alterthe amino acid sequences by amino acid substitutions. As discussedherein, amino acid substitutions can be made to alter the affinity ofthe CDRs for the protein (e.g. TIGIT or PVRIG, including both increasingand decreasing binding), as well as to alter additional functionalproperties of the antibodies. For example, the antibodies may beengineered to include modifications within the Fc region, typically toalter one or more functional properties of the antibody, such as serumhalf-life, complement fixation, Fc receptor binding, and/orantigen-dependent cellular cytotoxicity. Furthermore, an antibodyaccording to at least some embodiments of the invention may bechemically modified (e.g., one or more chemical moieties can be attachedto the antibody) or be modified to alter its glycosylation, again toalter one or more functional properties of the antibody. Suchembodiments are described further below. The numbering of residues inthe Fc region is that of the EU index of Kabat.

In one embodiment, the hinge region of Cm is modified such that thenumber of cysteine residues in the hinge region is altered, e.g.,increased or decreased. This approach is described further in U.S. Pat.No. 5,677,425 by Bodmer et al. The number of cysteine residues in thehinge region of CH1 is altered to, for example, facilitate assembly ofthe light and heavy chains or to increase or decrease the stability ofthe antibody.

In still another embodiment, the antibody can be modified to abrogate invivo Fab arm exchange, in particular when IgG4 constant domains areused. Specifically, this process involves the exchange of IgG4half-molecules (one heavy chain plus one light chain) between other IgG4antibodies that effectively results in bispecific antibodies which arefunctionally monovalent. Mutations to the hinge region and constantdomains of the heavy chain can abrogate this exchange (see Aalberse, RC, Schuurman J., 2002, Immunology 105:9-19). As outlined herein, amutation that finds particular use in the present invention is the S241Pin the context of an IgG4 constant domain. IgG4 finds use in the presentinvention as it has no significant effector function, and is thus usedto block the receptor binding to its ligand without cell depletion (e.g.PVRIG to PVRL2 or TIGIT to PVR).

In some embodiments, amino acid substitutions can be made in the Fcregion, in general for altering binding to FcγR receptors. By “Fc gammareceptor”, “FcγR” or “FcgammaR” as used herein is meant any member ofthe family of proteins that bind the IgG antibody Fc region and isencoded by an FcγR gene. In humans this family includes but is notlimited to FcγRI (CD64), including isoforms FcγRIa, FcγRIb, and FcγRIc;FcγRII (CD32), including isoforms FcγRIIa (including allotypes H131 andR131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; andFcγRIII (CD16), including isoforms FcγRIIIa (including allotypes V158and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 andFcγRIIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65, entirelyincorporated by reference), as well as any undiscovered human FcγRs orFcγR isoforms or allotypes. An FcγR may be from any organism, includingbut not limited to humans, mice, rats, rabbits, and monkeys. Mouse FcγRsinclude but are not limited to FcγRI (CD64), FcγRII (CD32), FcγRIII-1(CD16), and FcγRIII-2 (CD16-2), as well as any undiscovered mouse FcγRsor FcγR isoforms or allotypes.

There are a number of useful Fc substitutions that can be made to alterbinding to one or more of the FcγR receptors. Substitutions that resultin increased binding as well as decreased binding can be useful. Forexample, it is known that increased binding to FcγRIIIa generallyresults in increased ADCC (antibody dependent cell-mediatedcytotoxicity; the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause lysis of the target cell. Similarly, decreasedbinding to FcγRIIb (an inhibitory receptor) can be beneficial as well insome circumstances. Amino acid substitutions that find use in thepresent invention include those listed in U.S. Ser. No. 11/124,620(particularly FIG. 41) and U.S. Pat. No. 6,737,056, both of which areexpressly incorporated herein by reference in their entirety andspecifically for the variants disclosed therein.

In yet another example, the Fc region is modified to increase theability of the antibody to mediate antibody dependent cellularcytotoxicity (ADCC) and/or to increase the affinity of the antibody foran Fcγ receptor, and/or increase FcRn binding, by modifying one or moreamino acids at the following positions: 238, 239, 248, 249, 252, 254,255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285,286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309,312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337,338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430,434, 435, 437, 438 or 439. This approach is described further in PCTPublication WO 00/42072 by Presta. Moreover, the binding sites on humanIgG1 for FcγRI, FcγRII, FcγRIII and FcRn have been mapped and variantswith improved binding have been described (see Shields, R. L. et al.(2001) J Biol. Chem. 276:6591-6604). Specific mutations at positions256, 290, 298, 333, 334 and 339 are shown to improve binding to FcγRIII.Additionally, the following combination mutants are shown to improveFcγRIII binding: T256A/S298A, S298A/E333A, S298A/K224A andS298A/E333A/K334A. Furthermore, mutations such as M252Y/S254T/T256E orM428L/N434S improve binding to FcRn and increase antibody circulationhalf-life (see Chan C A and Carter P J (2010) Nature Rev Immunol10:301-316).

In addition, the antibodies of the invention are modified to increaseits biological half-life. Various approaches are possible. For example,one or more of the following mutations can be introduced: T252L, T254S,T256F, as described in U.S. Pat. No. 6,277,375 to Ward. Alternatively,to increase the biological half-life, the antibody can be altered withinthe C_(H1) or C_(L) region to contain a salvage receptor binding epitopetaken from two loops of a CH2 domain of an Fc region of an IgG, asdescribed in U.S. Pat. Nos. 5,869,046 and 6,121,022 by Presta et al.Additional mutations to increase serum half-life are disclosed in U.S.Pat. Nos. 8,883,973, 6,737,056 and 7,371,826 and include 428L, 434A,434S, and 428L/434S.

In still another embodiment, the glycosylation of an antibody ismodified. For example, an aglycosylated antibody can be made (i.e., theantibody lacks glycosylation). Glycosylation can be altered to, forexample, increase the affinity of the antibody for antigen or reduceeffector function such as ADCC. Such carbohydrate modifications can beaccomplished by, for example, altering one or more sites ofglycosylation within the antibody sequence, for example N297. Forexample, one or more amino acid substitutions can be made that result inelimination of one or more variable region framework glycosylation sitesto thereby eliminate glycosylation at that site, with an alaninereplacement finding use in some embodiments.

Additionally or alternatively, an antibody can be made that has analtered type of glycosylation, such as a hypofucosylated antibody havingreduced amounts of fucosyl residues or an antibody having increasedbisecting GlcNac structures. Such altered glycosylation patterns havebeen demonstrated to increase the ADCC ability of antibodies. Suchcarbohydrate modifications can be accomplished by, for example,expressing the antibody in a host cell with altered glycosylationmachinery. Cells with altered glycosylation machinery have beendescribed in the art and can be used as host cells in which to expressrecombinant antibodies according to at least some embodiments of theinvention to thereby produce an antibody with altered glycosylation. Seefor example, U.S. Patent Publication No. 20040110704 and WO 2003/035835.

Another modification of the antibodies herein that is contemplated bythe invention is PEGylation or the addition of other water solublemoieties, typically polymers, e.g., in order to enhance half-life. Anantibody can be PEGylated to, for example, increase the biological(e.g., serum) half-life of the antibody as is known in the art.

In addition to substitutions made to alter binding affinity to FcγRsand/or FcRn and/or increase in vivo serum half-life, additional antibodymodifications can be made, as described in further detail below.

In some cases, affinity maturation is done. Amino acid modifications inthe CDRs are sometimes referred to as “affinity maturation”. An“affinity matured” antibody is one having one or more alteration(s) inone or more CDRs which results in an improvement in the affinity of theantibody for antigen, compared to a parent antibody which does notpossess those alteration(s). In some cases, it may be desirable todecrease the affinity of an antibody to its antigen.

In some embodiments, one or more amino acid modifications are made inone or more of the CDRs of the antibodies of the invention (PVRIG orTIGIT antibodies). In general, only 1 or 2 or 3-amino acids aresubstituted in any single CDR, and generally no more than from 1, 2, 3.4, 5, 6, 7, 8 9 or 10 changes are made within a set of 6 CDRs (e.g.vhCDR1-3 and vlCDR1-3). However, it should be appreciated that anycombination of no substitutions, 1, 2 or 3 substitutions in any CDR canbe independently and optionally combined with any other substitution.

Affinity maturation can be done to increase the binding affinity of theantibody for the antigen by at least about 10% to 50-100-150% or more,or from 1 to 5 fold as compared to the “parent” antibody. Preferredaffinity matured antibodies will have nanomolar or even picomolaraffinities for the antigen. Affinity matured antibodies are produced byknown procedures. The correlation of affinity and efficacy is discussedbelow.

Alternatively, amino acid modifications can be made in one or more ofthe CDRs of the antibodies of the invention that are “silent”, e.g. thatdo not significantly alter the affinity of the antibody for the antigen.These can be made for a number of reasons, including optimizingexpression (as can be done for the nucleic acids encoding the antibodiesof the invention).

Thus, included within the definition of the CDRs and antibodies of theinvention are variant CDRs and antibodies; that is, the antibodies ofthe invention can include amino acid modifications in one or more of theCDRs of the enumerated antibodies of the invention. In addition, asoutlined below, amino acid modifications can also independently andoptionally be made in any region outside the CDRs, including frameworkand constant regions.

a. Generation of Additional Antibodies

Additional antibodies to human PVRIG can be done as is well known in theart, using well known methods such as those outlined in the examples.Thus, additional anti-PVRIG antibodies can be generated by traditionalmethods such as immunizing mice (sometimes using DNA immunization, forexample, such as is used by Aldevron), followed by screening againsthuman PVRIG protein and hybridoma generation, with antibody purificationand recovery.

VII. TIGIT ANTIBODIES OF THE INVENTION

The present invention provides anti-TIGIT antibodies. (For convenience,“anti-TIGIT antibodies” and “TIGIT antibodies” are usedinterchangeably). The anti-TIGIT antibodies of the inventionspecifically bind to human TIGIT, and preferably the ECD of human TIGIT.The invention further provides antigen binding domains, including fulllength antibodies, which contain a number of specific, enumerated setsof 6 CDRs, that bind to TIGIT.

Specific binding for TIGIT or a TIGIT epitope can be exhibited, forexample, by an antibody having a K_(D) of at least about 10⁻⁴ M, atleast about 10⁻⁵ M, at least about 10⁻⁶ M, at least about 10⁻⁷M, atleast about 10⁻⁸M, at least about 10⁻⁹M, alternatively at least about10⁻¹⁰ M, at least about 10⁻¹¹ M, at least about 10⁻¹² M, at least about10⁻¹³ M, at least about 10⁻¹⁴ M, at least about 10⁻¹⁵ M, or greater,where K_(D) refers to the equilibrium dissociation constant of aparticular antibody-antigen interaction. Typically, an antibody thatspecifically binds an antigen will have a K_(D) that is 20-, 50-, 100-,500-, 1000-, 5,000-, 10,000- or more times greater for a controlmolecule relative to the TIGIT antigen or epitope.

However, for optimal binding to TIGIT expressed on the surface of NK andT-cells, the antibodies preferably have a KD less 50 nM and mostpreferably less than 1 nM, with less than 0.1 nM and less than 1 pMfinding use in the methods of the invention

Also, specific binding for a particular antigen or an epitope can beexhibited, for example, by an antibody having a ka (referring to theassociation rate constant) for a TIGIT antigen or epitope of at least20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- or more times greater forthe epitope relative to a control, where ka refers to the associationrate constant of a particular antibody-antigen interaction.

In some embodiments, the anti-TIGIT antibodies of the invention bind tohuman TIGIT with a K_(D) of 100 nM or less, 50 nM or less, 10 nM orless, or 1 nM or less (that is, higher binding affinity), or 1 pM orless, wherein K_(D) is determined by known methods, e.g. surface plasmonresonance (SPR, e.g. Biacore assays), ELISA, KINEXA, and most typicallySPR at 25° or 37° C.

The TIGIT antibodies described herein are labeled as follows. Theantibodies have reference numbers, for example “CPA.9.086”. Thisrepresents the combination of the variable heavy and variable lightchains, as depicted in FIG. 53, for example, with the understanding thatthese antibodies include two heavy chains and two light chains.“CPA.9.086.VH” refers to the variable heavy portion of CPA. 9. 086,while “CPA. 9. 086.VL” is the variable light chain. “CPA. 9.086.vhCDR1”, “CPA. 9. 086.vhCDR2”, “CPA. 9. 086.vhCDR3”, “CPA. 9.086.vlCDR1”, “CPA. 9. 086.vlCDR2”, and “CPA. 9. 086.vlCDR3”, refers tothe CDRs are indicated. “CPA. 9. 086.HC” refers to the entire heavychain (e.g. variable and constant domain) of this molecule, and “CPA. 9.086.LC” refers to the entire light chain (e.g. variable and constantdomain) of the same molecule. In general, the human kappa light chain isused for the constant domain of each phage (or humanized hybridoma)antibody herein, although in some embodiments the lambda light constantdomain is used. “CPA. 9. 086.H1” refers to a full length antibodycomprising the variable heavy and light domains, including the constantdomain of Human IgG1 (hence, the H1; IgG1, IgG2, IgG3 and IgG4 sequencesare shown in FIG. 50). Accordingly, “CPA. 9. 086.H2” would be the CPA.9. 086 variable domains linked to a Human IgG2. “CPA. 9. 086.H3” wouldbe the CPA. 9. 086 variable domains linked to a Human IgG3, and “CPA. 9.086.H4” would be the CPA. 9. 086 variable domains linked to a HumanIgG4. Note that in some cases, the human IgGs may have additionalmutations, such are described below, and this can be annotated. Forexample, in many embodiments, there may be a S241P mutation in the humanIgG4, and this can be annotated as “CPA.9.086.H4(S241P)” for example.The human IgG4 sequence with this S241P hinge variant is shown in FIG.50. Other potential variants are IgG1(N297A), (or other variants thatablate glycosylation at this site and thus many of the effectorfunctions associated with FcγRIIIa binding), and IgG1(D265A), whichreduces binding to FcγR receptors.

The invention further provides variable heavy and light domains as wellas full length heavy and light chains.

In some embodiments, the invention provides scFvs that bind to TIGITcomprising a variable heavy domain and a variable light domain linked byan scFv linker as outlined above. The VL and VH domains can be in eitherorientation, e.g. from N- to C-terminus “VH-linker-VL” or “VL-linker”VH”. These are named by their component parts; for example, “scFv-CPA.9.086.VH-linker-VL” or “scFv-CPA.9.086.VL-linker-VH.” Thus,“scFv-CPA.9.086” can be in either orientation.

In many embodiments, the antibodies of the invention are human (derivedfrom phage) and block binding of TIGIT and PVR. As shown in FIGS. 58 and75, the CPA antibodies that both bind and block the receptor-ligandinteraction are as below, with their components outlined as well (asdiscussed in the “Sequence” section, the sequences of all but the scFvconstructs are in the sequence listing):

CPA.9.018, CPA.9.018.VH, CPA.9.018.VL, CPA.9.018.HC, CPA.9.018.LC,CPA.9.018.H1, CPA.9.018.H2, CPA.9.018.H3, CPA.9.018.H4;CPA.9.018.H4(S241P); CPA.9.018.vhCDR1, CPA.9.018.vhCDR2,CPA.9.018.vhCDR3, CPA.9.018.vlCDR1, CPA.9.018.vlCDR2, CPA.9.018.vlCDR3and scFv-CPA.9.018;

CPA.9.027, CPA.9.027.VH, CPA.9.027.VL, CPA.9.027.HC, CPA.9.027.LC,CPA.9.027.H1, CPA.9.027.H2, CPA.9.027.H3, CPA.9.027.H4;CPA.9.018.H4(S241P); CPA.9.027.vhCDR1, CPA.9.027.vhCDR2,CPA.9.027.vhCDR3, CPA.9.027.vlCDR1, CPA.9.027.vlCDR2, CPA.9.027.vlCDR3and scFv-CPA.9.027;

CPA.9.049, CPA.9.049.VH, CPA.9.049.VL, CPA.9.049.HC, CPA.9.049.LC,CPA.9.049.H1, CPA.9.049.H2, CPA.9.049.H3; CPA.9.049.H4;CPA.9.049.H4(S241P); CPA.9.049.vhCDR1, CPA.9.049.vhCDR2,CPA.9.049.vhCDR3, CPA.9.049.vlCDR1, CPA.9.049.vlCDR2, CPA.9.049.vlCDR3and scFv-CPA.9.049;

CPA.9.057, CPA.9.057.VH, CPA.9.057.VL, CPA.9.057.HC, CPA.9.057.LC,CPA.9.057.H1, CPA.9.057.H2, CPA.9.057.H3; CPA.9.057.H4;CPA.9.057.H4(S241P); CPA.9.057.vhCDR1, CPA.9.057.vhCDR2,CPA.9.057.vhCDR3, CPA.9.057.vlCDR1, CPA.9.057.vlCDR2, CPA.9.057.vlCDR3and scFv-CPA.9.057;

CPA.9.059, CPA.9.059.VH, CPA.9.059.VL, CPA.9.059.HC, CPA.9.059.LC,CPA.9.059.H1, CPA.9.059.H2, CPA.9.059.H3; CPA.9.059.H4;CPA.9.059.H4(S241P); CPA.9.059.vhCDR1, CPA.9.059.vhCDR2,CPA.9.059.vhCDR3, CPA.9.059.vlCDR1, CPA.9.059.vlCDR2, CPA.9.059.vlCDR3and scFv-CPA.9.059;

CPA.9.083, CPA.9.083.VH, CPA.9.083.VL, CPA.9.083.HC, CPA.9.083.LC,CPA.9.083.H1, CPA.9.083.H2, CPA.9.083.H3; CPA.9.083.H4;CPA.9.083.H4(S241P); CPA.9.083.vhCDR1, CPA.9.083.vhCDR2,CPA.9.083.vhCDR3, CPA.9.083.vlCDR1, CPA.9.083.vlCDR2, CPA.9.083.vlCDR3and scFv-CPA.9.083;

CPA.9.086, CPA.9.086.VH, CPA.9.086.VL, CPA.9.086.HC, CPA.9.086.LC,CPA.9.086.H1, CPA.9.086.H2, CPA.9.086.H3; CPA.9.086.H4;CPA.9.086.H4(S241P); CPA.9.086.vhCDR1, CPA.9.086.vhCDR2,CPA.9.086.vhCDR3, CPA.9.086.vlCDR1, CPA.9.086.vlCDR2, CPA.9.086.vlCDR3and scFv-CPA.9.086;

CPA.9.089, CPA.9.089.VH, CPA.9.089.VL, CPA.9.089.HC, CPA.9.089.LC,CPA.9.089.H1, CPA.9.089.H2, CPA.9.089.H3; CPA.9.089.H4;CPA.9.089.H4(S241P); CPA.9.089.vhCDR1, CPA.9.089.vhCDR2,CPA.9.089.vhCDR3, CPA.9.089.vlCDR1, CPA.9.089.vlCDR2, CPA.9.089.vlCDR3and scFv-CPA.9.089;

CPA.9.093, CPA.9.093.VH, CPA.9.093.VL, CPA.9.093.HC, CPA.9.093.LC,CPA.9.093.H1, CPA.9.093.H2, CPA.9.093.H3; CPA.9.093.H4;CPA.9.093.H4(S241P); CPA.9.093.vhCDR1, CPA.9.093.vhCDR2,CPA.9.093.vhCDR3, CPA.9.093.vlCDR1, CPA.9.093.vlCDR2, CPA.9.093.vlCDR3and scFv-CPA.9.093;

CPA.9.101, CPA.9.101.VH, CPA.9.101.VL, CPA.9.101.HC, CPA.9.101.LC,CPA.9.101.H1, CPA.9.101.H2, CPA.9.101.H3; CPA.9.101.H4;CPA.9.101.H4(S241P); CPA.9.101.vhCDR1, CPA.9.101.vhCDR2,CPA.9.101.vhCDR3, CPA.9.101.vlCDR1, CPA.9.101.vlCDR2, CPA.9.101.vlCDR3and scFv-CPA.9.101; and

CPA.9.103, CPA.9.103.VH, CPA.9.103.VL, CPA.9.103.HC, CPA.9.103.LC,CPA.9.103.H1, CPA.9.103.H2, CPA.9.103.H3; CPA.9.103.H4;CPA.9.103.H4(S241P); CPA.9.103.vhCDR1, CPA.9.103.vhCDR2,CPA.9.103.vhCDR3, CPA.9.103.vlCDR1, CPA.9.103.vlCDR2, CPA.9.103.vlCDR3and scFv-CPA.9.103.

Furthermore, the present invention provides a number of CHA antibodies,which are murine antibodies generated from hybridomas. As is well knownthe art, the six CDRs are useful when put into either human frameworkvariable heavy and variable light regions or when the variable heavy andlight domains are humanized.

Accordingly, the present invention provides antibodies, usually fulllength or scFv domains, that comprise the following sets of CDRs, thesequences of which are shown in FIG. 53 and/or the sequence listing:

CHA.9.536.1, CHA.9.536.1.VH, CHA.9.536.1.VL, CHA.9.536.1.HC,CHA.9.536.1.LC, CHA.9.536.1.H1, CHA.9.536.1.H2, CHA.9.536.1.H3;CHA.9.536.1.H4, CHA.9.536.1.H4(S241P), CHA.9.536.1.vhCDR1,CHA.9.536.1.vhCDR2, CHA.9.536.1.vhCDR3, CHA.9.536.1.vlCDR1,CHA.9.536.1.vlCDR2 and CHA.9.536.1.vhCDR3;

CHA.9.536.3, CHA.9.536.3.VH, CHA.9.536.3.VL, CHA.9.536.3.HC,CHA.9.536.3.LC, CHA.9.536.3.H1, CHA.9.536.3.H2, CHA.9.536.3.H3;CHA.9.536.3.H4, CHA.9.536.3.H4(S241P); CHA.9.536.3.vhCDR1,CHA.9.536.3.vhCDR2, CHA.9.536.3.vhCDR3, CHA.9.536.3.vlCDR1,CHA.9.536.3.vlCDR2 and CHA.9.536.3.vhCDR3;

CHA.9.536.4, CHA.9.536.4.VH, CHA.9.536.4.VL, CHA.9.536.4.HC,CHA.9.536.4.LC, CHA.9.536.4.H1, CHA.9.536.4.H2, CHA.9.536.4.H3;CHA.9.536.4.H4, CHA.9.536.4.H4(S241P), CHA.9.536.4.vhCDR1,CHA.9.536.4.vhCDR2, CHA.9.536.4.vhCDR3, CHA.9.536.4.vlCDR1,CHA.9.536.4.vlCDR2 and CHA.9.536.4.vhCDR3;

CHA.9.536.5, CHA.9.536.5.VH, CHA.9.536.5.VL, CHA.9.536.5.HC,CHA.9.536.5.LC, CHA.9.536.5.H1, CHA.9.536.5.H2, CHA.9.536.5.H3;CHA.9.536.5.H4, CHA.9.536.5.H4(S241P), CHA.9.536.5.vhCDR1,CHA.9.536.5.vhCDR2, CHA.9.536.5.vhCDR3, CHA.9.536.5.vlCDR1,CHA.9.536.5.vlCDR2 and CHA.9.536.5.vhCDR3;

CHA.9.536.6, CHA.9.536.6.VH, CHA.9.536.6.VL, CHA.9.536.6.HC,CHA.9.536.6.LC, CHA.9.536.6.H1, CHA.9.536.6.H2, CHA.9.536.6.H3;CHA.9.536.6.H4, CHA.9.536.6.vhCDR1, CHA.9.536.6.vhCDR2,CHA.9.536.6.vhCDR3, CHA.9.536.6.vlCDR1, CHA.9.536.6.vlCDR2 andCHA.9.536.6.vhCDR3;

CHA.9.536.7, CHA.9.536.7.VH, CHA.9.536.7.VL, CHA.9.536.7.HC,CHA.9.536.7.LC, CHA.9.536.7.H1, CHA.9.536.7.H2, CHA.9.536.7.H3;CHA.9.536.7.H4, CHA.9.536.5.H4(S241P); CHA.9.536.7.vhCDR1,CHA.9.536.7.vhCDR2, CHA.9.536.7.vhCDR3, CHA.9.536.7.vlCDR1,CHA.9.536.7.vlCDR2 and CHA.9.536.7.vhCDR3;

CHA.9.536.8, CHA.9.536.8.VH, CHA.9.536.8.VL, CHA.9.536.8.HC,CHA.9.536.8.LC, CHA.9.536.8.H1, CHA.9.536.8.H2, CHA.9.536.8.H3;CHA.9.536.8.H4, CHA.9.536.8.H4(S241P), CHA.9.536.8.vhCDR1,CHA.9.536.8.vhCDR2, CHA.9.536.8.vhCDR3, CHA.9.536.8.vlCDR1,CHA.9.536.8.vlCDR2 and CHA.9.536.8.vhCDR3;

CHA.9.560.1, CHA. 9.560.1VH, CHA. 9.560.1.VL, CHA. 9.560.1.HC, CHA.9.560.1.LC, CHA. 9.560.1.H1, CHA. 9.560.1.H2, CHA. 9.560.1.H3; CHA.9.560.1.H4, CHA. 9.560.1.H4(S241P), CHA. 9.560.1.vhCDR1, CHA.9.560.1.vhCDR2, CHA. 9.560.1.vhCDR3, CHA. 9.560.1.vlCDR1, CHA.9.560.1.vlCDR2 and CHA. 9.560.1.vhCDR3;

CHA.9.560.3, CHA. 9.560. 3VH, CHA. 9.560. 3.VL, CHA. 9.560. 3.HC, CHA.9.560. 3.LC, CHA. 9.560. 3.H1, CHA. 9.560. 3.H2, CHA. 9.560. 3.H3;CHA.9.560.3.H4, CHA.9.560.3.H4(S241P); CHA. 9.560. 3.vhCDR1, CHA. 9.560.3.vhCDR2, CHA. 9.560. 3.vhCDR3, CHA. 9.560. 3.vlCDR1, CHA. 9.560.3.vlCDR2 and CHA. 9.560. 3.vhCDR3;

CHA.9.560.4, CHA. 9.560. 4VH, CHA. 9.560. 4.VL, CHA. 9.560. 4.HC, CHA.9.560. 4.LC, CHA. 9.560. 4.H1, CHA. 9.560. 4.H2, CHA. 9.560. 4.H3;CHA.9.560.4.H4, CHA.9.560.4.H4(S241P), CHA. 9.560. 4.vhCDR1, CHA. 9.560.4.vhCDR2, CHA. 9.560. 4.vhCDR3, CHA. 9.560. 4.vlCDR1, CHA. 9.560.4.vlCDR2 and CHA. 9.560. 4.vhCDR3;

CHA.9.560.5, CHA. 9.560. SVH, CHA. 9.560. 5.VL, CHA. 9.560. 5.HC, CHA.9.560. 5.LC, CHA. 9.560. 5.H1, CHA. 9.560. 5.H2, CHA. 9.560. 5.H3; CHA.9.560. 5.H4, CHA. 9.560. 5.vhCDR1, CHA. 9.560. 5.vhCDR2, CHA. 9.560.5.vhCDR3, CHA. 9.560. 5.vlCDR1, CHA. 9.560. 5.vlCDR2 and CHA. 9.560.5.vhCDR3;

CHA.9.560.6, CHA. 9.560. 6VH, CHA. 9.560. 6.VL, CHA. 9.560. 6.HC, CHA.9.560. 6.LC, CHA. 9.560. 6.H1, CHA. 9.560. 6.H2, CHA. 9.560. 6.H3;CHA.9.560.6.H4, CHA.9.560.6.H4(S241P), CHA. 9.560. 6.vhCDR1, CHA. 9.560.6.vhCDR2, CHA. 9.560. 6.vhCDR3, CHA. 9.560. 6.vlCDR1, CHA. 9.560.6.vlCDR2 and CHA. 9.560. 6.vhCDR3;

CHA.9.560.7, CHA. 9.560. 7VH, CHA. 9.560. 7.VL, CHA. 9.560. 7.HC, CHA.9.560. 7.LC, CHA. 9.560. 7.H1, CHA. 9.560. 7.H2, CHA. 9.560. 7.H3;CHA.9.560.7.H4; CHA.9.560.7.H4(S241P); CHA. 9.560. 7.vhCDR1, CHA. 9.560.7.vhCDR2, CHA. 9.560. 7.vhCDR3, CHA. 9.560. 7.vlCDR1, CHA. 9.560.7.vlCDR2 and CHA. 9.560. 7.vhCDR3;

CHA.9.560.8, CHA. 9.560. 8VH, CHA. 9.560. 8.VL, CHA. 9.560. 8.HC, CHA.9.560. 8.LC, CHA. 9.560. 8.H1, CHA. 9.560. 8.H2, CHA. 9.560. 8.H3;CHA.9.560.8.H4, CHA.9.560.8.H4(S241P); CHA. 9.560. 8.vhCDR1, CHA. 9.560.8.vhCDR2, CHA. 9.560. 8.vhCDR3, CHA. 9.560. 8.vlCDR1, CHA. 9.560.8.vlCDR2 and CHA. 9.560. 8.vhCDR3;

CHA.9.546.1, CHA. 9. 546.1VH, CHA. 9. 546.1.VL, CHA. 9. 546.1.HC, CHA.9. 546.1.LC, CHA. 9. 546.1.H1, CHA. 9. 546.1.H2, CHA. 9. 546.1.H3;CHA.9.546.1.H4, CHA.9.546.1.H4(S241P), CHA. 9. 546.1.vhCDR1, CHA. 9.546.1.vhCDR2, CHA. 9. 546.1.vhCDR3, CHA. 9. 546.1.vlCDR1, CHA. 9.546.1.vlCDR2 and CHA. 9. 546.1.vhCDR3;

CHA.9.547.1, CHA. 9. 547.1VH, CHA. 9. 547.1.VL, CHA. 9. 547.1.HC, CHA.9. 547.1.LC, CHA. 9. 547.1.H1, CHA. 9. 547.1.H2, CHA. 9. 547.1.H3;CHA.9.547.1.H4, CHA.9.547.1.H4(S241P), CHA. 9. 547.1.vhCDR1, CHA. 9.547.1.vhCDR2, CHA. 9. 547.1.vhCDR3, CHA. 9. 547.1.vlCDR1, CHA. 9.547.1.vlCDR2 and CHA. 9. 547.1.vhCDR3;

CHA.9.547.2, CHA. 9. 547. 2VH, CHA. 9. 547. 2.VL, CHA. 9. 547. 2.HC,CHA. 9. 547. 2.LC, CHA. 9. 547. 2.H1, CHA. 9. 547. 2.H2, CHA. 9. 547.2.H3; CHA.9.547.2.H4, CHA.9.547.2.H4(S241P), CHA. 9. 547. 2.vhCDR1, CHA.9. 547. 2.vhCDR2, CHA. 9. 547. 2.vhCDR3, CHA. 9. 547. 2.vlCDR1, CHA. 9.547. 2.vlCDR2 and CHA. 9. 547. 2.vhCDR3;

CHA.9.547.3, CHA. 9. 547. 3VH, CHA. 9. 547. 3.VL, CHA. 9. 547. 3.HC,CHA. 9. 547. 3.LC, CHA. 9. 547. 3.H1, CHA. 9. 547. 3.H2, CHA. 9. 547.3.H3; CHA.9.547.3.H4, CHA.9.547.3.H4(S241P), CHA. 9. 547. 3.vhCDR1, CHA.9.547. 3.vhCDR2, CHA. 9. 547. 3.vhCDR3, CHA. 9. 547. 3.vlCDR1, CHA. 9.547. 3.vlCDR2 and CHA. 9. 547. 3.vhCDR3;

CHA.9.547.4, CHA. 9. 547. 4VH, CHA. 9. 547. 4.VL, CHA. 9. 547. 4.HC,CHA. 9.547. 4.LC, CHA. 9. 547. 4.H1, CHA. 9. 547. 4.H2, CHA. 9. 547.4.H3; CHA.9.547.4.H4, CHA.9.547.4.H4(S241P), CHA. 9. 547. 4.vhCDR1, CHA.9. 547. 4.vhCDR2, CHA. 9. 547. 4.vhCDR3, CHA. 9. 547. 4.vlCDR1, CHA. 9.547. 4.vlCDR2 and CHA. 9. 547. 4.vhCDR3;

CHA.9.547.6, CHA. 9. 547. 6 VH, CHA. 9. 547. 6.VL, CHA. 9. 547. 6.HC,CHA. 9. 547. 6.LC, CHA. 9. 547. 6.H1, CHA. 9. 547. 6.H2, CHA. 9. 547.6.H3; CHA.9.547.6.H4, CHA.9.547.6.H4(S241P), CHA. 9. 547. 6.vhCDR1, CHA.9. 547. 6.vhCDR2, CHA. 9. 547. 6.vhCDR3, CHA. 9. 547. 6.vlCDR1, CHA. 9.547. 6.vlCDR2 and CHA. 9. 547. 6.vhCDR3;

CHA.9.547.7, CHA. 9. 547. 7VH, CHA. 9. 547. 7.VL, CHA. 9. 547. 7.HC,CHA. 9. 547. 7.LC, CHA. 9. 547. 7.H1, CHA. 9. 547. 7.H2, CHA. 9. 547.7.H3; CHA.9.547.7.H4, CHA.9.547.7.H4(S241P), CHA. 9. 547. 7.vhCDR1, CHA.9. 547. 7.vhCDR2, CHA. 9. 547. 7.vhCDR3, CHA. 9. 547. 7.vlCDR1, CHA. 9.547. 7.vlCDR2 and CHA. 9. 547. 7.vhCDR3;

CHA.9.547.8, CHA. 9. 547. 8VH, CHA. 9. 547. 8.VL, CHA. 9. 547. 8.HC,CHA.9.547.8.LC, CHA. 9. 547. 8.H1, CHA. 9. 547. 8.H2, CHA. 9. 547. 8.H3;CHA.9.547.8.H4, CHA.9.547.8.H4(S241P), CHA. 9. 547. 8.vhCDR1, CHA. 9.547. 8.vhCDR2, CHA. 9. 547. 8.vhCDR3, CHA. 9. 547. 8.vlCDR1, CHA. 9.547. 8.vlCDR2 and CHA. 9. 547. 8.vhCDR3;

CHA.9.547.9, CHA.9.547.9, CHA.9.547.9VH, CHA.9.547.9.VL, CHA.9.547.9.HC, CHA.9.547.9.LC, CHA.9.547.9.H1, CHA.9.547.9.H2,CHA.9.547.9.H3; CHA.9.547.9.H4, CHA.9.547.9.H4, CHA.9.547.9.H4(S241P),CHA.9.547.9.H4(S241P), CHA.9.547.9.vhCDR1, CHA.9.547.9.vhCDR2,CHA.9.547.9.vhCDR3, CHA.9.547.9.vlCDR1, CHA.9.547.9.vlCDR2 andCHA.9.547.9.vhCDR3;

CHA.9.547.13, CHA.9.547.13, CHA.9.547. 13VH, CHA.9. 547.13.VL, CHA.9.547.13.HC, CHA. 9.547.13.LC, CHA. 9.547.13.H1, CHA.9.547.13.H2, CHA.9.547.13.H3; CHA.9.547.13.H4, CHA.9.547.13.H4, CHA.9.547.13.H4(S241P),CHA.9.547.13.H4(S241P), CHA. 9. 547.13.vhCDR1, CHA.9.547.13.vhCDR2,CHA.9.547. 13.vhCDR3, CHA. 9. 547.13.vlCDR1, CHA. 9. 547.13.vlCDR2 andCHA. 9. 547. 13. vhCDR3;

CHA.9.541.1, CHA. 9. 541.1.VH, CHA. 9. 541.1.VL, CHA. 9. 541.1.HC, CHA.9. 541.1.LC, CHA. 9. 541.1.H1, CHA. 9. 541.1.H2, CHA. 9. 541.1.H3;CHA.9.541.1.H4, CHA.9.541.1.H4(S241P), CHA. 9. 541.1.vhCDR1, CHA. 9.541.1.vhCDR2, CHA. 9. 541.1.vhCDR3, CHA. 9. 541.1.vlCDR1, CHA. 9.541.1.vlCDR2 and CHA. 9.541.1.vhCDR3;

CHA.9.541.3, CHA. 9. 541. 3.VH, CHA. 9. 541. 3.VL, CHA. 9. 541. 3.HC,CHA. 9. 541. 3.LC, CHA. 9. 541. 3.H1, CHA. 9. 541. 3.H2, CHA. 9. 541.3.H3; CHA.9.541.3.H4, CHA.9.541.3.H4(S241P), CHA. 9. 541. 3.vhCDR1, CHA.9. 541. 3.vhCDR2, CHA. 9. 541. 3.vhCDR3, CHA. 9. 541. 3.vlCDR1, CHA. 9.541. 3.vlCDR2 and CHA. 9.541. 3.vhCDR3;

CHA.9.541.4, CHA. 9. 541.4.VH, CHA. 9. 541. 4.VL, CHA. 9. 541. 4.HC,CHA. 9. 541. 4.LC, CHA. 9. 541. 4.H1, CHA. 9. 541. 4.H2, CHA. 9. 541.4.H3; CHA.9.541.4.H4, CHA.9.541.4.H4(S241P), CHA. 9. 541. 4.vhCDR1, CHA.9. 541. 4.vhCDR2, CHA. 9. 541. 4.vhCDR3, CHA. 9. 541. 4.vlCDR1, CHA. 9.541. 4.vlCDR2 and CHA. 9.541. 4.vhCDR3;

CHA.9.541.5, CHA. 9. 541. 5.VH, CHA. 9. 541. 5.VL, CHA. 9. 541. 5.HC,CHA. 9. 541. 5.LC, CHA. 9. 541. 5.H1, CHA. 9. 541. 5.H2, CHA. 9. 541.5.H3; CHA.9.541.5.H4, CHA.9.541.5.H4(S241P), CHA. 9. 541. 5.vhCDR1, CHA.9. 541. 5.vhCDR2, CHA. 9. 541. 5.vhCDR3, CHA. 9. 541. 5.vlCDR1, CHA. 9.541. 5.vlCDR2 and CHA. 9.541. 5.vhCDR3;

CHA.9.541.6, CHA. 9. 541. 6.VH, CHA. 9. 541. 6.VL, CHA. 9. 541. 6.HC,CHA. 9. 541. 6.LC, CHA. 9. 541. 6.H1, CHA. 9. 541. 6.H2, CHA. 9.541.6.H3; CHA.9.541.6.H4, CHA.9.541.6.H4(S241P), CHA. 9. 541. 6.vhCDR1,CHA. 9. 541. 6.vhCDR2, CHA. 9. 541. 6.vhCDR3, CHA. 9. 541. 6.vlCDR1,CHA. 9. 541. 6.vlCDR2 and CHA. 9.541. 6.vhCDR3;

CHA.9.541.7, CHA. 9. 541. 7.VH, CHA. 9. 541. 7.VL, CHA. 9. 541. 7.HC,CHA. 9. 541. 7.LC, CHA. 9. 541. 7.H1, CHA. 9. 541. 7.H2, CHA. 9. 541.7.H3; CHA.9.541.7.H4, CHA.9.541.7.H4(S241P), CHA. 9. 541. 7.vhCDR1, CHA.9. 541. 7.vhCDR2, CHA. 9. 541. 7.vhCDR3, CHA. 9. 541. 7.vlCDR1, CHA. 9.541. 7.vlCDR2 and CHA. 9.541. 7.vhCDR3; and

CHA.9.541.8, CHA. 9. 541. 8.VH, CHA. 9. 541. 8.VL, CHA. 9. 541. 8.HC,CHA. 9. 541. 8.LC, CHA. 9. 541. 8.H1, CHA. 9. 541. 8.H2, CHA. 9. 541.8.H3; CHA.9.541.8.H4, CHA.9.541.8.H4(S241P); CHA. 9. 541. 8vhCDR1, CHA.9. 541. 8.vhCDR2, CHA. 9. 541. 8.vhCDR3, CHA. 9. 541. 8.vlCDR1, CHA. 9.541. 8.vlCDR2 and CHA. 9.541. 8.vhCDR3.

In the case of scFvs comprising the CDRs of the antibodies above, theseare labeled as scFvs that include a scFv comprising a variable heavydomain with the vhCDRs, a linker and a variable light domain with thevlCDRs, again as above in either orientation. Thus the inventionincludes scFv-CHA.9.536.3.1, scFv-CHA.9.536.3, scFv-CHA.9.536.4,scFv-CHA.9.536.5, scFv-CHA.9.536.7, scFv-CHA.9.536.8, scFv-CHA.9.560.1,scFv-CHA.9.560.3, scFv-CHA.9.560.4, scFv-CHA.9.560.5, scFv-CHA.9.560.6,scFv-CHA.9. 560.7, scFv-CHA. 9.560. 8, scFv-CHA.9.546.1, scFv-CHA.9.547.1, scFv-CHA.9. 547.2, scFv-CHA. 9.547.3, scFv-CHA.9.547.4,scFv-CHA.9. 547.6, scFv-CHA.9. 547.7, scFv-CHA. 9.547. 8,scFv-CHA.9.547. 9, scFv-CHA.9. 547.13, scFv-CHA.9.541.1,scFv-CHA.9.541.3, scFv-CHA.9.541.4, scFv-CHA.9.541.5, scFv-CHA.9.541.6,scFv-CHA.9.541.7 and scFv-CHA.9.541.8.

In addition, CHA.9.543 binds to TIGIT but does not block the TIGIT-PVRinteraction.

As discussed herein, the invention further provides variants of theabove components (CPA and CHA), including variants in the CDRs, asoutlined above. Thus, the invention provides antibodies comprising a setof 6 CDRs as outlined herein that can contain one, two or three aminoacid differences in the set of CDRs, as long as the antibody still bindsto TIGIT. Suitable assays for testing whether an anti-TIGIT antibodythat contains mutations as compared to the CDR sequences outlined hereinare known in the art, such as Biacore assays.

In addition, the invention further provides variants of the abovevariable heavy and light chains. In this case, the variable heavy chainscan be 80%, 90%, 95%, 98% or 99% identical to the “VH” sequences herein,and/or contain from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid changes, ormore, when Fc variants are used. Variable light chains are provided thatcan be 80%, 90%, 95%, 98% or 99% identical to the “VL” sequences herein(and in particular CPA.9.086), and/or contain from 1, 2, 3, 4, 5, 6, 7,8, 9, 10 amino acid changes, or more, when Fc variants are used. Inthese embodiments, the invention includes these variants as long as theantibody still binds to TIGIT. Suitable assays for testing whether ananti-TIGIT antibody that contains mutations as compared to the CDRsequences outlined herein are known in the art, such as Biacore assays.

Similarly, heavy and light chains are provided that are 80%, 90%, 95%,98% or 99% identical to the full length “HC” and “LC” sequences herein(and in particular CPA.9.086), and/or contain from 1, 2, 3, 4, 5, 6, 7,8, 9, 10 amino acid changes, or more, when Fc variants are used. Inthese embodiments, the invention includes these variants as long as theantibody still binds to TIGIT. Suitable assays for testing whether ananti-TIGIT antibody that contains mutations as compared to the CDRsequences outlined herein are known in the art, such as Biacore assays.

In addition, the framework regions of the variable heavy and variablelight chains of either the CPA or CHA antibodies herein can be humanized(or, in the case of the CHA antibodies, “rehumanized”, to the extentthat alternative humanization methods can be done) as is known in theart (with occasional variants generated in the CDRs as needed), and thushumanized variants of the VH and VL chains of FIG. 53 can be generated(and in particular CPA.9.086). Furthermore, the humanized variable heavyand light domains can then be fused with human constant regions, such asthe constant regions from IgG1, IgG2, IgG3 and IgG4 (includingIgG4(S241P)).

In particular, as is known in the art, murine VH and VL chains can behumanized as is known in the art, for example, using the IgBLAST programof the NCBI website, as outlined in Ye et al. Nucleic Acids Res.41:W34-W40 (2013), herein incorporated by reference in its entirety forthe humanization methods. IgBLAST takes a murine VH and/or VL sequenceand compares it to a library of known human germline sequences. As shownherein, for the humanized sequences generated herein, the databases usedwere IMGT human VH genes (F+ORF, 273 germline sequences) and IMGT humanVL kappa genes (F+ORF, 74 germline sequences). An exemplary five CHAsequences were chosen: CHA.9.536, CHA9.560, CHA.9.546, CHA.9.547 andCHA.9.541 (see FIG. 53). For this embodiment of the humanization, humangermline IGHV1-46(allelel) was chosen for all 5 as the acceptor sequenceand the human heavy chain IGHJ4(allelel) joining region (J gene). Forthree of four (CHA.7.518, CHA.7.530, CHA.7.538_1 and CHA.7.538_2), humangermline IGKV1-39(allele 1) was chosen as the acceptor sequence andhuman light chain IGKJ2(allelel) (J gene) was chosen. The J gene waschosen from human joining region sequences compiled at IMGT® theinternational ImMunoGeneTics information system as www.imgt.org. CDRswere defined according to the AbM definition (seewww.bioinfo.org.uk/abs/).

In some embodiments, the anti-TIGIT antibodies of the present inventioninclude anti-TIGIT antibodies wherein the V_(H) and V_(L) sequences ofdifferent anti-TIGIT antibodies can be “mixed and matched” to createother anti-TIGIT antibodies. TIGIT binding of such “mixed and matched”antibodies can be tested using the binding assays described above. e.g.,ELISAs or Biacore assays). In some embodiments, when V_(H) and V_(L)chains are mixed and matched, a V_(H) sequence from a particularV_(H)/V_(L) pairing is replaced with a structurally similar V_(H)sequence. Likewise, in some embodiments, a V_(L) sequence from aparticular V_(H)/V_(L) pairing is replaced with a structurally similarV_(L) sequence. For example, the V_(H) and V_(L) sequences of homologousantibodies are particularly amenable for mixing and matching.

Accordingly, the TIGIT antibodies of the invention comprise CDR aminoacid sequences selected from the group consisting of (a) sequences aslisted herein; (b) sequences that differ from those CDR amino acidsequences specified in (a) by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or moreamino acid substitutions; (c) amino acid sequences having 90% orgreater, 95% or greater, 98% or greater, or 99% or greater sequenceidentity to the sequences specified in (a) or (b); (d) a polypeptidehaving an amino acid sequence encoded by a polynucleotide having anucleic acid sequence encoding the amino acids as listed herein. Inparticular, the CPA.9.086 antibody can have sequences selected from (a),(b), (c) or (d).

Additionally included in the definition of TIGIT antibodies areantibodies that share identity to the TIGIT antibodies enumeratedherein. That is, in certain embodiments, an anti-TIGIT antibodyaccording to the invention comprises heavy and light chain variableregions comprising amino acid sequences that are identical to all orpart of the anti-TIGIT amino acid sequences of preferred anti-TIGITantibodies, respectively, wherein the antibodies retain the desiredfunctional properties of the parent anti-TIGIT antibodies. The percentidentity between the two sequences is a function of the number ofidentical positions shared by the sequences (i.e., % homology=# ofidentical positions/total # of positions×100), taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences. The comparison of sequencesand determination of percent identity between two sequences can beaccomplished using a mathematical algorithm, as described in thenon-limiting examples below.

The percent identity between two amino acid sequences can be determinedusing the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci.,4:11-17 (1988)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4. In addition, the percent identity betweentwo amino acid sequences can be determined using the Needleman andWunsch (I Mol. Biol. 48:444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availablecommercially), using either a Blossum 62 matrix or a PAM250 matrix, anda gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2,3, 4, 5, or 6.

Additionally or alternatively, the protein sequences of the presentinvention can further be used as a “query sequence” to perform a searchagainst public databases to, for example, identify related sequences.Such searches can be performed using the XBLAST program (version 2.0) ofAltschul, et al. (1990) J Mol. Biol. 215:403-10. BLAST protein searchescan be performed with the XBLAST program, score=50, wordlength=3 toobtain amino acid sequences homologous to the antibody moleculesaccording to at least some embodiments of the invention. To obtaingapped alignments for comparison purposes, Gapped BLAST can be utilizedas described in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)can be used.

In general, the percentage identity for comparison between TIGITantibodies is at least 75%, at least 80%, at least 90%, with at leastabout 95, 96, 97, 98 or 99% percent identity being preferred. Thepercentage identity may be along the whole amino acid sequence, forexample the entire heavy or light chain or along a portion of thechains. For example, included within the definition of the anti-TIGITantibodies of the invention are those that share identity along theentire variable region (for example, where the identity is 95 or 98%identical along the variable regions), or along the entire constantregion, or along just the Fc domain. In particular, the inventionprovides TIGIT antibodies that have at least 75%, at least 80%, at least90%, with at least about 95, 96, 97, 98 or 99% percent identity beingpreferred, with the CPA.9.086 antibody.

In addition, also included are sequences that may have the identicalCDRs but changes in the framework portions of the variable domain (orentire heavy or light chain). For example, TIGIT antibodies includethose with CDRs identical to those shown in FIG. 53 but whose identityalong the variable region can be lower, for example 95 or 98% percentidentical. In particular, the invention provides TIGIT antibodies thathave identical CDRs to CPA.9.086 but with framework regions that are 95or 98% identical to CPA.9.086.

A. TIGIT Antibodies that Compete for Binding

The present invention provides not only the enumerated antibodies butadditional antibodies that compete with the enumerated antibodies (theCPA numbers enumerated herein that specifically bind to TIGIT) tospecifically bind to the TIGIT molecule. As is shown in Example 16, theTIGIT antibodies of the invention “bin” into different epitope bins.Among the 44 TIGIT antibodies in the epitope binning study, there arefour communities, each having related pairwise blocking patterns, whichseparate into 12 total discrete bins outlined herein and shown in FIGS.67 and 68. There are twelve discrete bins outlined herein; 1) BM9-H4,CHA.9.525, CPA.9.081-H4, CHA.9.538, CHA.9.553, CPA.9.069-H4, CHA.9.543,CHA.9.556, CPA.9.077-H4 and CHA.9.561; 2) CHA.9.560 and CHA.9.528; 3)CHA.9.552, CHA.9.521, CHA.9.541, CHA.9.529, CHA.9.519, CHA.9.527 andCHA.9.549; 4) CPA.9.057-H4 and CHA.9.554; 5) CHA.9.546, CPA.9.012-H4,CHA.9.547, CPA.9.013-H4, CPA.9.018-H4, MBSA43-M1, Sino PVR-Fc(ligand),CHA.9.555, PVR-Fc M2A(ligand), BM29-H4, CPA.9.027-H4, CPA.9.049-H4 andCPA.9.053-H4; 6) CPA.9.064-H4; 7) BM26-H4; 8) CPA.9.059-H4; 9) CHA.9.535and CPA.9.009-H4; 10) CHA.9.536, CHA.9.522 and CPA.9.015-H4; 11)CPA.9.011-H4 and BM8-H4 and 12) CPA.9.071-H4.

Thus, the invention provides anti-TIGIT antibodies that compete forbinding with antibodies that are in discrete epitope bins 1 to 12. In aparticular embodiment, the invention provides anti-TIGIT antibodies thatcompete for binding with CPA.9.086 and are at least 95, 96, 97, 98 or99% identical to CPA.9.086.

Additional antibodies that compete with the enumerated antibodies aregenerated, as is known in the art and generally outlined below.Competitive binding studies can be done as is known in the art,generally using SPR/Biacore® binding assays, as well as ELISA andcell-based assays.

VIII. PVRIG ANTIBODIES

The present invention provides anti-PVRIG antibodies. (For convenience,“anti-PVRIG antibodies” and “PVRIG antibodies” are usedinterchangeably). The anti-PVRIG antibodies of the inventionspecifically bind to human PVRIG, and preferably the ECD of human PVRIG.

Specific binding for PVRIG or a PVRIG epitope can be exhibited, forexample, by an antibody having a KD of at least about 10⁴ M, at leastabout 10⁻⁵ M, at least about 10⁻⁶ M, at least about 10⁻⁷ M, at leastabout 10⁻⁸ M, at least about 10⁻⁹M, alternatively at least about 10⁻¹⁰M, at least about 10⁻¹¹ M, at least about 10⁻¹²M, or greater, where KDrefers to a dissociation rate of a particular antibody-antigeninteraction. Typically, an antibody that specifically binds an antigenwill have a KD that is 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- ormore times greater for a control molecule relative to the PVRIG antigenor epitope.

However, as shown in the Examples of WO2016/134333, for optimal bindingto PVRIG expressed on the surface of NK and T-cells, the antibodiespreferably have a KD less 50 nM and most preferably less than 1 nM, withless than 0.1 nM and less than 1 pM and 0.1 pM finding use in themethods of the invention.

Also, specific binding for a particular antigen or an epitope can beexhibited, for example, by an antibody having a KA or Ka for a PVRIGantigen or epitope of at least 20-, 50-, 100-, 500-, 1000-, 5,000-,10,000- or more times greater for the epitope relative to a control,where KA or Ka refers to an association rate of a particularantibody-antigen interaction.

In some embodiments, the anti-PVRIG antibodies of the invention bind tohuman PVRIG with a K_(D) of 100 nM or less, 50 nM or less, 10 nM orless, or 1 nM or less (that is, higher binding affinity), or 1 pM orless, wherein K_(D) is determined by known methods, e.g. surface plasmonresonance (SPR, e.g. Biacore assays), ELISA, KINEXA, and most typicallySPR at 25° or 37° C.

It is important to note that binding affinity for the anti-PVRIGantibodies is surprisingly correlated with activity. A cumulativeanalysis of screening data shows that the affinity of the anti-PVRIGantibodies of the invention correlated highly with their ability to bindto primary human T cells. More specifically, the antibodies that gavethe highest maximum signal on T cells were those with affinities in thepicomolar range. Antibodies that had affinities in the low nanomolarrange and above gave relatively weak maximum signals on T cells. Thus,the data indicates that the usefulness of anti-PVRIG antibodies for Tcell-based immunotherapy can likely be defined, in part, based on theiraffinity. Reference is made to antibody sequences from WO2016/134333,hereby incorporated by reference and in particular for the anti-PVRIGantigen binding domains outlined in FIG. 38 (depicting sequences thatbind PVRIG and block the interaction of PVRIG and PVRL2), FIG. 39(depicting sequences that bind PVRIG and do not block the interaction ofPVRIG and PVRL2), FIG. 40 (depicting CDRs and data from theseantibodies), and FIG. 41 (depicting CDRs from hybridomas that bind andblock). That is, the Figures and Legends as well as the particularsequences and SEQ ID NO:s from all CPA.7 and CHA.7 antibodies (includingCDRs, VH and VL and full length sequences) from WO2016/134333 areexpressly incorporated herein.

FIG. 45 illustrates the ability of two anti-PVRIG antibodies ofdifferent affinities to bind primary CD8 T cells. As shown in FIG. 45,CHA.7.518 has approximately an 8-fold higher affinity than CPA.7.021(sequence in WO2016/13433) as measured by binding to HEK cellsengineered to over-express PVRIG (HEK hPVRIG). Consistent with this,CHA.7.518 has approximately a 13-fold higher affinity than CPA.7.021 asmeasured by binding to Jurkat cells. The higher affinity of CHA.7.518did correspond to a greater maximum binding signal from HEK hPVRIGcells, but not Jurkat cells.

In contrast, CHA.7.518 consistently gave a higher maximum binding signalfrom primary CD8 T cells, as compared to CPA.7.021. This is illustratedin a binding titration experiment where different concentrations ofisotype or anti-PVRIG antibodies were added to primary CD8 T cells, andthe resultant maximum binding signal measured. In the two donorsillustrated (FIG. 45), CHA.7.518 consistently gave a higher maximumsignal (geometric mean fluorescence intensity, gMFI) than CPA.7.021 in atitration dependent manner. gMFIr=geometric fluorescence intensity ofthe antibody of interest/geometric fluorescence intensity of the controlantibody. The gMFIr measures the signal the antibody of interest givesrelative to an isotype antibody at a fixed concentration of both.

Accordingly, the anti-PVRIG antibodies of the invention have bindingaffinities (as measured using techniques outlined herein) in thepicomolar range, e.g. from 0.1 to 9 pM, with from about 0.2 to about 2being preferred, and from about 0.2 to about 0.5 being of particularuse.

As for the TIGIT antibodies, the PVRIG antibodies are similarly labeledas follows. The antibodies have reference numbers, for example“CHA.7.518.1”. This represents the combination of the variable heavy andvariable light chains, as depicted in FIG. 3 for example, with theunderstanding that these antibodies include two heavy chains and twolight chains. “CPA. 7.518.1.VH” refers to the variable heavy portion ofCPA. 7.518.1, while “CPA.7.518.1.VL” is the variable light chain. “CPA.7.518.1.vhCDR1”, “CPA.7.518.1.vhCDR2”, “CPA. 7.518.1.vhCDR3”, “CPA.7.518.1.vlCDR1”, “CPA. 7.518.1.vlCDR2”, and “CPA. 7.518.1.vlCDR3”,refers to the CDRs are indicated. “CPA. 7.518.1.HC” refers to the entireheavy chain (e.g. variable and constant domain) of this molecule, and“CPA. 7.518.1.LC” refers to the entire light chain (e.g. variable andconstant domain) of the same molecule. In general, the human kappa lightchain is used for the constant domain of each phage (or humanizedhybridoma) antibody herein, although in some embodiments the lambdalight constant domain is used. “CPA. 7.518.1.H1” refers to a full-lengthantibody comprising the variable heavy and light domains, including theconstant domain of Human IgG1 (hence, the H1; IgG1, IgG2, IgG3 and IgG4sequences are shown in FIG. 50). Accordingly, “CPA. 7.518.1.H2” would bethe CPA. 7.518.1 variable domains linked to a Human IgG2. “CPA.7.518.1.H3” would be the CPA. 7.518.1 variable domains linked to a HumanIgG3, and “CPA. 7.518.1.H4” would be the CPA. 7.518.1 variable domainslinked to a Human IgG4. Note that in some cases, the human IgGs may haveadditional mutations, such are described below, and this can beannotated. For example, in many embodiments, there may be a S241Pmutation in the human IgG4, and this can be annotated as “CPA.7.518.1.H4(S241P)” for example. The human IgG4 sequence with this S241Phinge variant is shown in FIG. 50. Other potential variants areIgG1(N297A), (or other variants that ablate glycosylation at this siteand thus many of the effector functions associated with FcγRIIIabinding), and IgG1(D265A), which reduces binding to FcγR receptors.

The invention further provides variable heavy and light domains as wellas full length heavy and light chains.

In some embodiments, the invention provides scFvs that bind to PVRIGcomprising a variable heavy domain and a variable light domain linked byan scFv linker as outlined above. The VL and VH domains can be in eitherorientation, e.g. from N- to C-terminus “VH-linker-VL” or “VL-linker”VH”. These are named by their component parts; for example,“scFv-CHA.7.518.1VH-linker-VL” or “scFv-CPA. 7.518.1.VL-linker-VH.”Thus, “scFv-CPA. 7.518.1” can be in either orientation.

IX. NUCLEIC ACIDS ENCODING ANTIBODIES

Nucleic acid compositions encoding the antibodies of the invention arealso provided, as well as expression vectors containing the nucleicacids and host cells transformed with the nucleic acid and/or expressionvector compositions. As will be appreciated by those in the art, theprotein sequences depicted herein can be encoded by any number ofpossible nucleic acid sequences, due to the degeneracy of the geneticcode.

The nucleic acid compositions that encode the antibodies will depend onthe format of the antibody. For traditional, tetrameric antibodiescontaining two heavy chains and two light chains are encoded by twodifferent nucleic acids, one encoding the heavy chain and one encodingthe light chain. These can be put into a single expression vector or twoexpression vectors, as is known in the art, transformed into host cells,where they are expressed to form the antibodies of the invention. Insome embodiments, for example when scFv constructs are used, a singlenucleic acid encoding the variable heavy chain-linker-variable lightchain is generally used, which can be inserted into an expression vectorfor transformation into host cells. The nucleic acids can be put intoexpression vectors that contain the appropriate transcriptional andtranslational control sequences, including, but not limited to, signaland secretion sequences, regulatory sequences, promoters, origins ofreplication, selection genes, etc.

Preferred mammalian host cells for expressing the recombinant antibodiesaccording to at least some embodiments of the invention include ChineseHamster Ovary (CHO cells), PER.C6, HEK293 and others as is known in theart.

The nucleic acids may be present in whole cells, in a cell lysate, or ina partially purified or substantially pure form. A nucleic acid is“isolated” or “rendered substantially pure” when purified away fromother cellular components or other contaminants, e.g., other cellularnucleic acids or proteins, by standard techniques, includingalkaline/SDS treatment, CsCl banding, column chromatography, agarose gelelectrophoresis and others well known in the art.

To create a scFv gene, the V_(H)- and V_(L)-encoding DNA fragments areoperatively linked to another fragment encoding a flexible linker, e.g.,encoding the amino acid sequence (Gly4-Ser)3 and others discussedherein, such that the V_(H) and V_(L) sequences can be expressed as acontiguous single-chain protein, with the V_(L) and V_(H) regions joinedby the flexible linker.

X. FORMULATIONS

The therapeutic compositions used in the practice of the foregoingmethods (and in particular CHA.7.518.1.H4(S241P) and CPA.9.086) can beformulated into pharmaceutical compositions comprising a carriersuitable for the desired delivery method. Suitable carriers include anymaterial that when combined with the therapeutic composition retains theanti-tumor function of the therapeutic composition and is generallynon-reactive with the patient's immune system. Examples include, but arenot limited to, any of a number of standard pharmaceutical carriers suchas sterile phosphate buffered saline solutions, bacteriostatic water,and the like (see, generally, Remington's Pharmaceutical Sciences 16thEdition, A. Osal., Ed., 1980). Acceptable carriers, excipients, orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed and may include buffers.

In a preferred embodiment, the pharmaceutical composition that comprisesthe antibodies of the invention may be in a water-soluble form, such asbeing present as pharmaceutically acceptable salts, which is meant toinclude both acid and base addition salts. “Pharmaceutically acceptableacid addition salt” refers to those salts that retain the biologicaleffectiveness of the free bases and that are not biologically orotherwise undesirable, formed with inorganic acids and the like.“Pharmaceutically acceptable base addition salts” include those derivedfrom inorganic bases and the like.

Administration of the pharmaceutical composition comprising antibodiesof the present invention, preferably in the form of a sterile aqueoussolution, may be done in a variety of ways, including, but not limitedto subcutaneously and intravenously.

The dosing amounts and frequencies of administration are, in a preferredembodiment, selected to be therapeutically or prophylacticallyeffective. As is known in the art, adjustments for protein degradation,systemic versus localized delivery, and rate of new protease synthesis,as well as the age, body weight, general health, sex, diet, time ofadministration, drug interaction and the severity of the condition maybe necessary, and will be ascertainable with routine experimentation bythose skilled in the art.

In order to treat a patient, a therapeutically effective dose of the Fcvariant of the present invention may be administered. By“therapeutically effective dose” herein is meant a dose that producesthe effects for which it is administered. The exact dose will depend onthe purpose of the treatment, and will be ascertainable by one skilledin the art using known techniques.

XI. METHODS FOR USING ANTIBODIES

The antibodies of the invention, including both PVRIG and TIGITantibodies, can be used in a number of diagnostic and therapeuticapplications. In some cases, the decision of which antibody toadminister to a patient is done using an evaluation of the expressionlevels (either gene expression levels or protein expression levels, withthe latter being preferred) of sample tumor biopsies to determinewhether the sample is overexpressing either TIGIT or PVRIG, or both, todetermine what therapeutic antibodies to administer.

A. Diagnostic Uses

Accordingly, the antibodies of the invention also find use in the invitro or in vivo diagnosis, including imaging, of tumors thatover-express either PVRIG or TIGIT, respectively. It should be noted,however, that as discussed herein, both TIGIT and PVRIG, asimmuno-oncology target proteins, are not necessarily overexpressed oncancer cells, but rather within the immune infiltrates in the cancer.Thus it is the mechanism of action, e.g. activation of immune cells suchas T cells and NK cells, that results in cancer diagnosis. Accordingly,these antibodies can be used to diagnose cancer. Diagnosis using PVRIGantibodies is also outlined in WO 2016/134333, [0434 to 0459], herebyincorporated by reference.

Generally, diagnosis can be done in several ways. In one embodiment, atissue from a patient, such as a biopsy sample, is contacted with aTIGIT antibody, generally labeled, such that the antibody binds to theendogenous TIGIT. The level of signal is compared to that of normalnon-cancerous tissue either from the same patient or a reference sample,to determine the presence or absence of cancer. The biopsy sample can befrom a solid tumor, a blood sample (for lymphomas and leukemias such asALL, T cell lymphoma, etc).

In general, in this embodiment, the anti-TIGIT is labeled, for examplewith a fluorophore or other optical label, that is detected using afluorometer or other optical detection system as is well known in theart. In an alternate embodiment, a secondary labeled antibody iscontacted with the sample, for example using an anti-human IgG antibodyfrom a different mammal (mouse, rat, rabbit, goat, etc.) to form asandwich assay as is known in the art. Alternatively, the anti-TIGIT mAbcould be directly labeled (i.e. biotin) and detection can be done by asecondary Ab directed to the labeling agent in the art.

Once over-expression of TIGIT is seen, treatment can proceed with theadministration of an anti-TIGIT antibody according to the invention asoutlined herein.

In other embodiments, in vivo diagnosis is done. Generally, in thisembodiment, the anti-TIGIT antibody (including antibody fragments) isinjected into the patient and imaging is done. In this embodiment, forexample, the antibody is generally labeled with an optical label or anMRI label, such as a gadolinium chelate, radioactive labeling of mAb(including fragments).

In some embodiments, the antibodies described herein are used for bothdiagnosis and treatment, or for diagnosis alone. When anti-TIGITantibodies are used for both diagnosis and treatment, some embodimentsrely on two different anti-TIGIT antibodies to two different epitopes,such that the diagnostic antibody does not compete for binding with thetherapeutic antibody, although in some cases the same antibody can beused for both. For example, this can be done using antibodies that arein different bins, e.g. that bind to different epitopes on TIGIT, suchas outlined herein. Thus included in the invention are compositionscomprising a diagnostic antibody and a therapeutic antibody, and in someembodiments, the diagnostic antibody is labeled as described herein. Inaddition, the composition of therapeutic and diagnostic antibodies canalso be co-administered with other drugs as outlined herein.

Particularly useful antibodies for use in diagnosis include, but are notlimited to these enumerated antibodies, or antibodies that utilize theCDRs with variant sequences, or those that compete for binding with anyof the antibodies in FIG. 53.

In many embodiments, a diagnostic antibody is labeled. By “labeled”herein is meant that the antibodies disclosed herein have one or moreelements, isotopes, or chemical compounds attached to enable thedetection in a screen or diagnostic procedure. In general, labels fallinto several classes: a) immune labels, which may be an epitopeincorporated as a fusion partner that is recognized by an antibody, b)isotopic labels, which may be radioactive or heavy isotopes, c) smallmolecule labels, which may include fluorescent and colorimetric dyes, ormolecules such as biotin that enable other labeling methods, and d)labels such as particles (including bubbles for ultrasound labeling) orparamagnetic labels that allow body imagining. Labels may beincorporated into the antibodies at any position and may be incorporatedin vitro or in vivo during protein expression, as is known in the art.

Diagnosis can be done either in vivo, by administration of a diagnosticantibody that allows whole body imaging as described below, or in vitro,on samples removed from a patient. “Sample” in this context includes anynumber of things, including, but not limited to, bodily fluids(including, but not limited to, blood, urine, serum, lymph, saliva, analand vaginal secretions, perspiration and semen), as well as tissuesamples such as result from biopsies of relevant tissues.

In addition, as outlined below and in the Examples and Figures,information regarding the protein expression levels of either PVRIG orTIGIT, or both, or PVRIG and PD-1, or TIGIT and PD-1, can be used todetermine which antibodies should be administered to a patient.

B. Cancer Treatment

The antibodies of the invention find particular use in the treatment ofcancer. In general, the antibodies of the invention areimmunomodulatory, in that rather than directly attack cancerous cells,the antibodies of the invention stimulate the immune system, generallyby inhibiting the action of the checkpoint receptor (e.g. PVRIG orTIGIT). Thus, unlike tumor-targeted therapies, which are aimed atinhibiting molecular pathways that are crucial for tumor growth anddevelopment, and/or depleting tumor cells, cancer immunotherapy is aimedto stimulate the patient's own immune system to eliminate cancer cells,providing long-lived tumor destruction. Various approaches can be usedin cancer immunotherapy, among them are therapeutic cancer vaccines toinduce tumor-specific T cell responses, and immunostimulatory antibodies(i.e. antagonists of inhibitory receptors=immune checkpoints) to removeimmunosuppressive pathways.

Clinical responses with targeted therapy or conventional anti-cancertherapies tend to be transient as cancer cells develop resistance, andtumor recurrence takes place. However, the clinical use of cancerimmunotherapy in the past few years has shown that this type of therapycan have durable clinical responses, showing dramatic impact on longterm survival. However, although responses are long term, only a smallnumber of patients respond (as opposed to conventional or targetedtherapy, where a large number of patients respond, but responses aretransient).

By the time a tumor is detected clinically, it has already evaded theimmune-defense system by acquiring immunoresistant and immunosuppressiveproperties and creating an immunosuppressive tumor microenvironmentthrough various mechanisms and a variety of immune cells.

Accordingly, the antibodies of the invention are useful in treatingcancer. Due to the nature of an immuno-oncology mechanism of action, thecheckpoint receptor (TIGIT or PVRIG) does not necessarily need to beoverexpressed on or correlated with a particular cancer type; that is,the goal is to have the antibodies de-suppress T cell and NK cellactivation, such that the immune system will go after the cancers.

“Cancer,” as used herein, refers broadly to any neoplastic disease(whether invasive or metastatic) characterized by abnormal anduncontrolled cell division causing malignant growth or tumor (e.g.,unregulated cell growth.) The term “cancer” or “cancerous” as usedherein should be understood to encompass any neoplastic disease (whetherinvasive, non-invasive or metastatic) which is characterized by abnormaland uncontrolled cell division causing malignant growth or tumor,non-limiting examples of which are described herein. This includes anyphysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer are exemplified in theworking examples and also are described within the specification.

Non-limiting examples of cancer that can be treated using the antibodiesof the invention include, but are not limited to, carcinoma, lymphoma,blastoma, sarcoma, and leukemia. More particular examples of suchcancers include squamous cell cancer, lung cancer (including small-celllung cancer, non-small cell lung cancer, adenocarcinoma of the lung, andsquamous carcinoma of the lung), cancer of the peritoneum,hepatocellular cancer, gastric or stomach cancer (includinggastrointestinal cancer), pancreatic cancer, glioblastoma, cervicalcancer, ovarian cancer, melanoma, non melanoma skin cancer (squamous andbasal cell carcinoma), liver cancer, bladder cancer, hepatoma, breastcancer, colon cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney or renal cancer, livercancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma and various types of head and neck cancer, as well as B-celllymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL);small lymphocytic (SL) NHL; intermediate grade/follicular NHL;intermediate grade diffuse NHL; high grade immunoblastic NHL; high gradelymphoblastic NHL; high grade small non-cleaved cell NHL; bulky diseaseNHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom'sMacroglobulinemia); chronic lymphocytic leukemia (CLL); acutelymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblasticleukemia; multiple myeloma and post-transplant lymphoproliferativedisorder (PTLD).

As shown in the Examples of WO2016/134333, PVRIG is over expressedand/or correlates with tumor lymphocyte infiltration (as demonstrated bycorrelation to CD3, CD4, CD8 and PD-1 expression) in a number ofdifferent tumors of various origins, and thus is useful in treating anycancer, including but not limited to, prostate cancer, liver cancer(HCC), colorectal cancer, ovarian cancer, endometrial cancer, breastcancer, pancreatic cancer, stomach cancer, cervical cancer, head andneck cancer, thyroid cancer, testis cancer, urothelial cancer, lungcancer, melanoma, non melanoma skin cancer (squamous and basal cellcarcinoma), glioma, renal cancer (RCC), lymphoma (non-Hodgkins' lymphoma(NHL) and Hodgkin's lymphoma (HD)), Acute myeloid leukemia (AML), T cellAcute Lymphoblastic Leukemia (T-ALL), Diffuse Large B cell lymphoma,testicular germ cell tumors, mesothelioma, and esophageal cancer.

In particular, CHA.7.518.1H4(S241P) finds use in treating prostatecancer, liver cancer (HCC), colorectal cancer, ovarian cancer,endometrial cancer, breast cancer, pancreatic cancer, stomach cancer,cervical cancer, head and neck cancer, thyroid cancer, testis cancer,urothelial cancer, lung cancer, melanoma, non melanoma skin cancer(squamous and basal cell carcinoma), glioma, renal cancer (RCC),lymphoma (NHL or HL), Acute myeloid leukemia (AML), T cell AcuteLymphoblastic Leukemia (T-ALL), Diffuse Large B cell lymphoma,testicular germ cell tumors, mesothelioma, bladder cancer and esophagealcancer.

In particular, CHA.7.538.1.2.H4(S241P) finds use in treating prostatecancer, liver cancer (HCC), colorectal cancer, ovarian cancer,endometrial cancer, breast cancer, pancreatic cancer, stomach cancer,cervical cancer, head and neck cancer, thyroid cancer, testis cancer,urothelial cancer, lung cancer, melanoma, non melanoma skin cancer(squamous and basal cell carcinoma), glioma, renal cancer (RCC),lymphoma (NHL or HL), Acute myeloid leukemia (AML), T cell AcuteLymphoblastic Leukemia (T-ALL), Diffuse Large B cell lymphoma,testicular germ cell tumors, mesothelioma, bladder cancer and esophagealcancer.

In particular, CPA.9.086H4(S241P) finds use in treating prostate cancer,liver cancer (HCC), colorectal cancer, ovarian cancer, endometrialcancer, breast cancer, pancreatic cancer, stomach cancer, cervicalcancer, head and neck cancer, thyroid cancer, testis cancer, urothelialcancer, lung cancer, melanoma, non melanoma skin cancer (squamous andbasal cell carcinoma), glioma, renal cancer (RCC), lymphoma (NHL or HL),Acute myeloid leukemia (AML), T cell Acute Lymphoblastic Leukemia(T-ALL), Diffuse Large B cell lymphoma, testicular germ cell tumors,mesothelioma, bladder cancer and esophageal cancer.

In particular CPA.9.083H4(S241P) finds use in treating prostate cancer,liver cancer (HCC), colorectal cancer, ovarian cancer, endometrialcancer, breast cancer, pancreatic cancer, stomach cancer, cervicalcancer, head and neck cancer, thyroid cancer, testis cancer, urothelialcancer, lung cancer, melanoma, non melanoma skin cancer (squamous andbasal cell carcinoma), glioma, renal cancer (RCC), lymphoma (NHL or HL),Acute myeloid leukemia (AML), T cell Acute Lymphoblastic Leukemia(T-ALL), Diffuse Large B cell lymphoma, testicular germ cell tumors,mesothelioma, bladder cancer and esophageal cancer.

In particular CHA.9.547.7.H4(S241P) finds use in treating prostatecancer, liver cancer (HCC), colorectal cancer, ovarian cancer,endometrial cancer, breast cancer, pancreatic cancer, stomach cancer,cervical cancer, head and neck cancer, thyroid cancer, testis cancer,urothelial cancer, lung cancer, melanoma, non melanoma skin cancer(squamous and basal cell carcinoma), glioma, renal cancer (RCC),lymphoma (NHL or HL), Acute myeloid leukemia (AML), T cell AcuteLymphoblastic Leukemia (T-ALL), Diffuse Large B cell lymphoma,testicular germ cell tumors, mesothelioma, bladder cancer and esophagealcancer.

In particular CHA.9.547.13.H4(S241P) finds use in treating prostatecancer, liver cancer (HCC), colorectal cancer, ovarian cancer,endometrial cancer, breast cancer, pancreatic cancer, stomach cancer,cervical cancer, head and neck cancer, thyroid cancer, testis cancer,urothelial cancer, lung cancer, melanoma, non melanoma skin cancer(squamous and basal cell carcinoma), glioma, renal cancer (RCC),lymphoma (NHL or HL), Acute myeloid leukemia (AML), T cell AcuteLymphoblastic Leukemia (T-ALL), Diffuse Large B cell lymphoma,testicular germ cell tumors, mesothelioma, bladder cancer and esophagealcancer.

C. TIGIT Antibody Monotherapy

The TIGIT antibodies of the invention find particular use in thetreatment of cancer as a monotherapy. Due to the nature of animmuno-oncology mechanism of action, TIGIT does not necessarily need tobe overexpressed on or correlated with a particular cancer type; thatis, the goal is to have the anti-TIGIT antibodies de-suppress T cell andNK cell activation, such that the immune system will go after thecancers.

While any anti-TIGIT antibody of FIG. 53 find us in the treatment ofcancer (including the activation of T cells as outlined below),CPA.9.086.H4(S241P), CPA.9.083.H4(S241P), CHA.9.547.7.H4(S241P), andCHA.9.547.13.H4(S241P), find particular use in some embodiments.

D. PVRIG Antibody Monotherapy

The PVRIG antibodies of the invention find particular use in thetreatment of cancer as a monotherapy. Due to the nature of animmuno-oncology mechanism of action, TIGIT does not necessarily need tobe overexpressed on or correlated with a particular cancer type; thatis, the goal is to have the anti-TIGIT antibodies de-suppress T cell andNK cell activation, such that the immune system will go after thecancers.

In particular, CHA.7.518.1H4(S241P) finds use as a monotherapy.

Similarly, in particular, CHA.7.538.1.2.H4(S241P) finds use as amonotherapy. in treating prostate cancer, liver cancer (HCC), colorectalcancer, ovarian cancer, endometrial cancer, breast cancer, pancreaticcancer, stomach cancer, cervical cancer, head and neck cancer, thyroidcancer, testis cancer, urothelial cancer, lung cancer, melanoma, nonmelanoma skin cancer (squamous and basal cell carcinoma), glioma, renalcancer (RCC), lymphoma (NHL or HL), Acute myeloid leukemia (AML), T cellAcute Lymphoblastic Leukemia (T-ALL), Diffuse Large B cell lymphoma,testicular germ cell tumors, mesothelioma, bladder cancer and esophagealcancer.

E. Combination Therapies

As is known in the art, combination therapies comprising a therapeuticantibody targeting an immunotherapy target and an additional therapeuticagent, specific for the disease condition, are showing great promise.For example, in the area of immunotherapy, there are a number ofpromising combination therapies using a chemotherapeutic agent (either asmall molecule drug or an anti-tumor antibody) or with animmuno-oncology antibody.

The terms “in combination with” and “co-administration” are not limitedto the administration of said prophylactic or therapeutic agents atexactly the same time. Instead, it is meant that the antibody and theother agent or agents are administered in a sequence and within a timeinterval such that they may act together to provide a benefit that isincreased versus treatment with only either the antibody of the presentinvention or the other agent or agents. It is preferred that theantibody and the other agent or agents act additively, and especiallypreferred that they act synergistically. Such molecules are suitablypresent in combination in amounts that are effective for the purposeintended. The skilled medical practitioner can determine empirically, orby considering the pharmacokinetics and modes of action of the agents,the appropriate dose or doses of each therapeutic agent, as well as theappropriate timings and methods of administration.

Accordingly, the antibodies of the present invention may be administeredconcomitantly with one or more other therapeutic regimens or agents. Theadditional therapeutic regimes or agents may be used to improve theefficacy or safety of the antibody. Also, the additional therapeuticregimes or agents may be used to treat the same disease or a comorbidityrather than to alter the action of the antibody. For example, anantibody of the present invention may be administered to the patientalong with chemotherapy, radiation therapy, or both chemotherapy andradiation therapy.

1. TIGIT Antibodies with Chemotherapeutic Small Molecules

The TIGIT antibodies of the present invention may be administered incombination with one or more other prophylactic or therapeutic agents,including but not limited to cytotoxic agents, chemotherapeutic agents,cytokines, growth inhibitory agents, anti-hormonal agents, kinaseinhibitors, anti-angiogenic agents, cardioprotectants, immunostimulatoryagents, immunosuppressive agents, agents that promote proliferation ofhematological cells, angiogenesis inhibitors, protein tyrosine kinase(PTK) inhibitors, or other therapeutic agents.

In this context, a “chemotherapeutic agent” is a chemical compounduseful in the treatment of cancer. Examples of chemotherapeutic agentsinclude alkylating agents such as thiotepa and cyclosphosphamide, alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines suchas benzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,triethylenephosphoramide, triethylenethiophosphoramide andtrimethylolomelamine; acetogenins (especially bullatacin andbullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL′);beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin(including the synthetic analogue topotecan (HYCAMTN®), CPT-11(irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including itsadozelesin, carzelesin and bizelesin synthetic analogues);podophyllotoxin; podophyllinic acid; teniposide; cryptophycins(particularly cryptophycin 1 and cryptophycin 8); dolastatin;duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1);eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosoureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;antibiotics such as the enediyne antibiotics (e. g., calicheamicin,especially calicheamicin gammall and calicheamicin omegall (see, e.g.,Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, includingdynemicin A; an esperamicin; as well as neocarzinostatin chromophore andrelated chromoprotein enediyne antiobiotic chromophores),aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins,dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, porfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS NaturalProducts, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium;tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine;trichothecenes (especially T-2 toxin, verracurin A, roridin A andanguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); thiotepa; taxoids, e.g., paclitaxel (TAXOL®;Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE®,cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), anddocetaxel (TAXOTERE®; Rhone-Poulenc Rorer, Antony, France);chloranbucil; gemcitabine (GEMZARM®); 6-thioguanine; mercaptopurine;methotrexate; platinum analogs such as cisplatin and carboplatin;vinblastine (VELBAN®); platinum; etoposide (VP-16); ifosfamide;mitoxantrone; vincristine (ONCOVIN®); oxaliplatin; leucovovin;vinorelbine (NAVELBINE®); novantrone; edatrexate; daunomycin;aminopterin; ibandronate; topoisomerase inhibitor RFS 2000;difluoromethylornithine (DMFO); retinoids such as retinoic acid;capecitabine (XELODA®); pharmaceutically acceptable salts, acids orderivatives of any of the above; as well as combinations of two or moreof the above such as CHOP, an abbreviation for a combined therapy ofcyclophosphamide, doxorubicin, vincristine, and prednisolone; CVP, anabbreviation for a combined therapy of cyclophosphamide, vincristine,and prednisolone; and FOLFOX, an abbreviation for a treatment regimenwith oxaliplatin (ELOXATIN®) combined with 5-FU and leucovorin.

According to at least some embodiments, the anti TIGIT immune moleculescould be used in combination with any of the known in the art standardof care cancer treatment (as can found, for example, the World Wide Webat cancer.gov/cancertopics).

Thus, in some cases, the anti-PVRIG antibodies outlined herein(particularly including CHA.7.538.1.2.H4(S241P) orCHA.7.518.1.H4(S241P)) can be combined with chemotherapeutic agents.Similarly, the anti-TIGIT antibodies outlined herein (particularlyincluding CPA.9.086H4(S241P), CPA.9.083H4(S241P) andCHA.9.547.13.H4(S241P)) can be combined with chemotherapeutic agents.

In addition, the anti-PVRIG and anti-TIGIT antibodies of the inventioncan also be administered with other checkpoint inhibitors or activators.

2. TIGIT and Checkpoint Antibody Combination Therapy

As shown herein, the TIGIT antibodies of the invention can be combinedwith one of a number of checkpoint receptor antibodies. In someembodiments, a patient's tumor may be evaluated for expression ofreceptors and the results then used to inform a clinician as to whichantibodies to administer: PVRIG and PD-1, TIGIT and PD-1 or TIGIT andPVRIG. These assays are described below.

a. Anti-TIGIT Antibodies in Combination with Anti-PD-1 Antibodies

In one embodiment, the invention provides combinations of the anti-TIGITantibodies of the invention and anti-PD-1 antibodies.

In one embodiment, a biopsy is taken from a tumor from a patient withcancer, and dissociated as is known in the art for FACS analysis. Thecells are stained with labeled antibodies to (1) TIGIT (for exampleusing any described herein or others in the art such as MBSA43); (2)PD-1 (for example using those known in the art including EH12.2H7,Keytruda®, Opdivo®, etc.); (3) PD-L1 (for example using those known inthe art such as BM-1 outlined herein) and (4) PVR (for example usingthose known in the art such as SKII.4); and (5) an isotype controlantibody. FACS is done, and for each receptor, the percentage of thecells expressing the receptor relative to the control antibody iscalculated. If the percentage of positive cells for TIGIT, PD-1, PD-1and PVR is ≥1% for all 4 receptors, then the patient is treated withantibodies to TIGIT and PD-1 as outlined herein.

Accordingly, the invention provides combinations of the anti-TIGITantibodies of the invention and anti-PD-1 antibodies. There are twoapproved anti-PD-1 antibodies, pembrolizumab (Keytruda®) and nivolumab(Opdivo®) and many more in development which can be used in combinationwith the anti-TIGIT antibodies of the invention.

Accordingly, the invention provides the specific combinations of:CPA.9.083.H4(S241P) (as shown in FIG. 53B) with pembrolizumab;CPA.9.083.H4(S241P) as shown in FIG. 53B with nivolumab;CPA.9.086.H4(S241P) as shown in FIG. 53A with pembrolizumab;CPA.9.086.H4(S241P) as shown in FIG. 53A with nivolumab;CHA.9.547.7H4(S241P) with pembrolizumab; CHA.9.547.7H4(S241P withnivolumab; CHA.9.547.13.H4(S241P) with pembrolizumab andCHA.9.547.13.H4(S241P) with nivolumab. (Reference is made to thesequence listing).

b. Anti-TIGIT Antibodies in Combination with Anti-CTLA-4 Antibodies

In another embodiment, the invention provides combinations of theanti-TIGIT antibodies of the invention and anti-CTLA-4 antibodies. Thereare two approved anti-CTLA-4 antibodies, ipilimumab (Yervoy®), andtremelimumab, as well as others in development, which can be used incombination with the anti-TIGIT antibodies of the invention.

Accordingly, the invention provides the specific combinations of:CPA.9.083.H4(S241P) with ipilimumab; CPA.9.083.H4(S241P) withtremelimumab; CPA.9.086.H4(S241P) with ipilimumab; CPA.9.086.H4(S241P)with tremelimumab; CHA.9.547.7H4(S241P) with ipilimumab;CHA.9.547.7H4(S241P) with tremelimumab; CHA.9.547.13.H4(S241P) withipilimumab and CHA.9.547.13.H4(S241P) with tremelimumab.

c. Anti-TIGIT Antibodies in Combination with Anti-PD-L1 Antibodies

In another embodiment, the invention provides combinations of theanti-TIGIT antibodies of the invention and anti-PD-L1 antibodies. Thereare three approved anti-PD-L1 antibodies, atezolizumab (TECENTRIQ®),avelumab (BAVENCIO®), and durvalumab (IMFINZI™), as well as otheranti-PD-L1 antibodies in development, which can be used in combinationwith the anti-TIGIT antibodies of the invention.

Accordingly, the invention provides the specific combinations of:CPA.9.083.H4(S241P) with atezolizumab; CPA.9.083.H4(S241P) withavelumab; CPA.9.083.H4(S241P) with durvalumab; CPA.9.086.H4(S241P) withatezolizumab; CPA.9.086.H4(S241P) with avelumab; CPA. 9.086.H4(S241P)with durvalumab; CHA.9.547.7H4(S241P) with atezolizumab;CHA.9.547.7H4(S241P) with avelumab; CHA.9.547.7H4(S241P) withdurvalumab; CHA.9.547.13.H4(S241P) with atezolizumab;CHA.9.547.13.H4(S241P) with avelumab; and CHA.9.547.13.H4(S241P) withdurvalumab.

d. Anti-TIGIT Antibodies in Combination with Anti-LAG-3 Antibodies

In another embodiment, the invention provides combinations of theanti-TIGIT antibodies of the invention and anti-LAG-3 antibodies. Thereare several anti-LAG-3 antibodies in development, including BMS-986016(see, International Patent Application No. WO2010/019570A2, incorporatedby reference herein in its entirety) GSK2831781 (see, US Patent Applic.No. 2016/0017037A, incorporated by reference herein in its entirety),and Merck clones 22D2, 11C9, 4A10, and/or 19E8 (see, WO2016/028672A1,incorporated by reference herein in its entirety) and GSK2831781 as wellas others in development, which can be used in combination with theanti-TIGIT antibodies of the invention.

Accordingly, the invention provides the specific combinations of:CPA.9.083.H4(S241P) with BMS-986016; CPA.9.083.H4(S241P) withGSK2831781; CPA.9.086.H4(S241P) with BMS-986016; CPA.9.086.H4(S241P)with GSK2831781; CHA.9.547.7H4(S241P) with BMS-986016;CHA.9.547.7H4(S241P) with GSK2831781; CHA.9.547.13.H4(S241P) withBMS-986016 and CHA.9.547.13.H4(S241P) with GSK2831781.

Accordingly, the invention also provides the specific combinations of:CPA.9.083.H4(S241P) with Merck clones 22D2, 11C9, and/or 4A10;CPA.9.086.H4(S241P) with Merck clones 22D2, 11C9, and/or 4A10;CHA.9.547.7H4(S241P) with Merck clones 22D2, 11C9, and/or 4A10;CHA.9.547.13.H4(S241P) with Merck clones 22D2, 11C9, and/or 4A10.

e. Anti-TIGIT Antibodies in Combination with Anti-TIM-3 Antibodies

In another embodiment, the invention provides combinations of theanti-TIGIT antibodies of the invention and anti-TIM-3 antibodies. Thereis at least one anti-TIM-3 antibody in development, TSR-022, as well asothers in development, which can be used in combination with theanti-TIGIT antibodies of the invention.

Accordingly, the invention provides the specific combinations of:CPA.9.083.H4(S241P) with TSR-022; CPA.9.086.H4(S241P with TSR-0226;CHA.9.547.7H4(S241P) with TSR-022; and CHA.9.547.13.H4(S241P) withTSR-022.

f. Anti-TIGIT Antibodies in Combination with Anti-BTLA Antibodies

In another embodiment, the invention provides combinations of theanti-TIGIT antibodies of the invention and anti-BTLA antibodies, seeWO2011/014438, hereby incorporated by reference in its entirety, andparticularly for the CDRs and full length sequences of the anti-BTLAantibodies disclosed therein. Accordingly, the invention provides thespecific combinations of: CPA.9.083.H4(S241P) with an anti-BTLAantibody; CPA.9.086.H4(S241P) with an anti-BTLA antibody;CHA.9.547.7H4(S241P) with an anti-BTLA antibody; andCHA.9.547.13.H4(S241P with an anti-BTLA antibody.

g. TIGIT Antibodies with Anti-Tumor Antibodies

In some embodiments, the anti-TIGIT antibodies of the invention areco-administered with antibodies that, unlike immuno-oncology/checkpointinhibitors that generally act on the immune system to increase apatient's native immune response, instead are directed against aspecific tumor target antigen (TTA). There are a wide number of anti-TTAantibodies either approved or in development that can be combined withthe present TIGIT antibodies. Currently approved antibodies, include,but are not limited to, cetuximab, panitumumab, nimotuzumab (all toEGFR), rituximab (CD20), trastuzumab and pertuzumab (HER2), alemtuzumab(CD52), bevacizumab (VEGF), ofatumumab (CD20), denosumab (RANK ligand),brentuximab (CD30), daratumumab (CD38), ibritumomab (CD20) andipilimumab (CTLA-4). Specific target oncology antibodies in clinicaltrials that can be combined with the anti-TIGIT antibodies hereininclude, but are not limited to, anti-CTLA4 mAbs, such as ipilimumab,tremelimumab; anti-PD-1 such as nivolumab BMS-936558/MDX-1106/ONO-4538,AMP224, CT-011, MK-3475, anti-PDL-1 antagonists such asBMS-936559/MDX-1105, MEDI4736, RG-7446/MPDL3280A; Anti-LAG-3 such asIMP-321), anti-TIM-3, anti-BTLA, anti-B7-H4, anti-B7-H3, Anti-VISTA;Agonistic antibodies targeting immunostimulatory proteins, includinganti-CD40 mAbs such as CP-870,893, lucatumumab, dacetuzumab; anti-CD137mAbs such as BMS-663513 urelumab (anti-4-IBB; see, for example, U.S.Pat. Nos. 7,288,638 and 8,962,804, incorporated by reference herein intheir entireties); PF-05082566 utomilumab (see, for example, U.S. Pat.Nos. 8,821,867; 8,337,850; and 9,468,678, as well as InternationalPatent Application Publication No. WO 2012/032433, incorporated byreference herein in their entireties); anti-OX40 mAbs, such as anti-OX40(see, for example, WO2006/029879 or WO2010096418, incorporated byreference herein in their entireties); anti-GITR mAbs such as TRX518(see, for example, U.S. Pat. No. 7,812,135, incorporated by referenceherein in its entirety); anti-CD27 mAbs, such as varlilumab CDX-1127(see, for example, WO 2016/145085 and U.S. Patent Publication Nos. US2011/0274685 and US 2012/0213771, incorporated by reference herein intheir entireties) anti-ICOS mAbs (for example, MEDI-570, JTX-2011, andanti-TIM3 antibodies (see, for example, WO 2013/006490 or U.S. PatentPublication No US 2016/0257758, incorporated by reference herein intheir entireties), as well as monoclonal antibodies to prostate cancer,ovarian cancer, breast cancer, endometrial cancer, multiple myeloma,melanoma, lymphomas, lung cancers including small cell lung cancer,kidney cancer, colorectal cancer, pancreatic cancer, gastric cancer,brain cancer, (see generally www.clinicaltrials.gov).

3. PVRIG and PD-1 Combination Therapy

As shown herein, the PVRIG antibodies of the invention can be combinedwith one of a number of checkpoint receptor antibodies.

a. Anti-PVRIG Antibodies in Combination with Anti-PD-1 Antibodies

In another embodiment, the invention provides combinations of theanti-PVRIG antibodies of the invention and anti-PD-1 antibodies.

In one embodiment, a biopsy is taken from a tumor from a patient withcancer, and dissociated as is known in the art for FACS analysis. Thecells are stained with labeled antibodies to (1) PVRIG (generally usingCHA.7.518.1H4(S241P), for example, although any outlined inWO2016/134333 (specifically including any that bind, even if they don'tblock) or WO2017/041004) can be used); (2) PD-1 (for example using thoseknown in the art including EH12.2H7, Keytruda®, Opdivo®, etc.); (3)PD-L1 (for example using those known in the art such as BM-1 outlinedherein) and (4) PVRL2 (for example using those known in the art such asTX11); and (5) an isotype control antibody. FACS is done, and for eachreceptor, the percentage of the cells expressing the receptor relativeto the control antibody is calculated. If the percentage of positivecells for PVRIG, PD-1, PD-1 and PVRL2 is ≥1% for all 4 receptors, thenthe patient is treated with antibodies to PVRIG and PD-1 as outlinedherein.

There are two approved anti-PD-1 antibodies, pembrolizumab (Keytruda®)and nivolumab (Opdivo®) and many more in development which can be usedin combination with the anti-PVRIG antibodies of the invention.

Accordingly, the invention provides the specific combinations of:CHA.7.518.1.H4(S241P) (as shown in FIG. 3) with pembrolizumab;CHA.7.518.1.H4(S241P) as shown in FIG. 3 with nivolumab;CHA.7.538.1.2.H4(S241P) as shown in FIG. 3 with pembrolizumab andCHA.7.538.1.2.H4(S241P) as shown in with nivolumab.

b. Anti-PVRIG Antibodies in Combination with Anti-CTLA-4 Antibodies

In another embodiment, the invention provides combinations of theanti-PVRIG antibodies of the invention and anti-CTLA-4 antibodies. Thereare two approved anti-CTLA-4 antibodies, ipilimumab (Yervoy®), andtremelimumab, as well as others in development, which can be used incombination with the anti-TIGIT antibodies of the invention.

Accordingly, the invention provides the specific combinations of:CHA.7.518.1.H4(S241P) with ipilimumab; CHA.7.518.1.H4(S241P) withtremelimumab; CHA.7.538.1.2.H4(S241P) with ipilimumab andCHA.7.538.1.2.H4(S241P) with tremelimumab.

c. Anti-PVRIG Antibodies in Combination with Anti-PD-L1 Antibodies

In another embodiment, the invention provides combinations of theanti-PVRIG antibodies of the invention and anti-PD-L1 antibodies. Thereare three approved anti-PD-L1 antibodies, atezolizumab (TECENTRIQ®),avelumab (BAVENCIO®), and durvalumab, as well as other anti-PD-L1antibodies in development, which can be used in combination with theanti-TIGIT antibodies of the invention.

Accordingly, the invention provides the specific combinations of:CHA.7.518.1.H4(S241P) with atezolizumab; CPA.7518.1.H4(S241P) withavelumab; CHA.7.518.1.H4(S241P) with durvalumab; CHA.7.538.1.2.H4(S241P)with atezolizumab; CHA.7.538.1.2.H4(S241P) with avelumab andCHA.7.538.1.2.H4(S241P) with durvalumab.

d. Anti-PVRIG Antibodies in Combination with Anti-LAG-3 Antibodies

In another embodiment, the invention provides combinations of theanti-PVRIG antibodies of the invention and anti-LAG-3 antibodies. Thereare several anti-LAG-3 antibodies in development, including BMS-986016(see, International Patent Application No. WO2010/019570A2, incorporatedby reference herein in its entirety) GSK2831781 (see, US Patent Applic.No. 2016/0017037A, incorporated by reference herein in its entirety),and Merck clones 22D2, 11C9, 4A10, and/or 19E8 (see, WO2016/028672A1,incorporated by reference herein in its entirety) and GSK2831781 as wellas others in development, which can be used in combination with theanti-PVRIG antibodies of the invention.

Accordingly, the invention provides the specific combinations of:CHA.7.518.1.H4(S241P) with BMS-986016; CHA.7.518.1.H4(S241P) withGSK2831781; CHA.7.538.1.2.H4(S241P) with BMS-986016 andCHA.7.538.1.2.H4(S241P) with GSK2831781.

Accordingly, the invention also provides the specific combinations of:CHA.7.518.1.H4(S241P) with Merck clones 22D2, 11C9, and/or 4A10 andCHA.7.538.1.2.H4(S241P) with Merck clones 22D2, 11C9, and/or 4A10.

e. Anti-PVRIG Antibodies in Combination with Anti-TIM-3 Antibodies

In another embodiment, the invention provides combinations of theanti-PVRIG antibodies of the invention and anti-TIM-3 antibodies. Thereis at least one anti-TIM-3 antibody in development, TSR-022, as well asothers in development, which can be used in combination with theanti-PVRIG antibodies of the invention.

Accordingly, the invention provides the specific combinations of:CHA.7.518.1.H4(S241P) with TSR-022 and CHA.7.538.1.2.H4(S241P) withTSR-0226.

f. Anti-PVRIG Antibodies in Combination with Anti-BTLA Antibodies

In another embodiment, the invention provides combinations of theanti-PVRIG antibodies of the invention and anti-BTLA antibodies, seeWO2011/014438, hereby incorporated by reference in its entirety, andparticularly for the CDRs and full length sequences of the anti-BTLAantibodies disclosed therein. Accordingly, the invention provides thespecific combinations of: CHA.7.518.1.H4(S241P) with an anti-BTLAantibody and CHA.7.538.1.2.H4(S241P) with an anti-BTLA antibody.

g. PVRIG Antibodies with Anti-Tumor Antibodies

In some embodiments, the anti-PVRIG antibodies of the invention areco-administered with antibodies that, unlike immuno-oncology/checkpointinhibitors that generally act on the immune system to increase apatient's native immune response, instead are directed against aspecific tumor target antigen (TTA). There are a wide number of anti-TTAantibodies either approved or in development that can be combined withthe present PVRIG antibodies, including CHA.7.518.1.H4(S241P) andCHA.7.538.1.2.H4(S241P). Currently approved antibodies, include, but arenot limited to, cetuximab, panitumumab, nimotuzumab (all to EGFR),rituximab (CD20), trastuzumab and pertuzumab (HER2), alemtuzumab (CD52),bevacizumab (VEGF), ofatumumab (CD20), denosumab (RANK ligand),brentuximab (CD30), daratumumab (CD38), ibritumomab (CD20) andipilimumab (CTLA-4). Specific target oncology antibodies in clinicaltrials that can be combined with the anti-PVRIG antibodies hereininclude, but are not limited to, anti-CTLA4 mAbs, such as ipilimumab,tremelimumab; anti-PD-1 such as nivolumab BMS-936558/MDX-1106/ONO-4538,AMP224, CT-011, MK-3475, anti-PDL-1 antagonists such asBMS-936559/MDX-1105, MEDI4736, RG-7446/MPDL3280A; Anti-LAG-3 such asIMP-321), anti-TIM-3, anti-BTLA, anti-B7-H4, anti-B7-H3, Anti-VISTA;Agonistic antibodies targeting immunostimulatory proteins, includinganti-CD40 mAbs such as CP-870,893, lucatumumab, dacetuzumab; anti-CD137mAbs such as BMS-663513 urelumab (anti-4-1BB; see, for example, U.S.Pat. Nos. 7,288,638 and 8,962,804, incorporated by reference herein intheir entireties); PF-05082566 utomilumab (see, for example, U.S. Pat.Nos. 8,821,867; 8,337,850; and 9,468,678, as well as InternationalPatent Application Publication No. WO 2012/032433, incorporated byreference herein in their entireties); anti-OX40 mAbs, such as anti-OX40(see, for example, WO2006/029879 or WO2010096418, incorporated byreference herein in their entireties); anti-GITR mAbs such as TRX518(see, for example, U.S. Pat. No. 7,812,135, incorporated by referenceherein in its entirety); anti-CD27 mAbs, such as varlilumab CDX-1127(see, for example, WO 2016/145085 and U.S. Patent Publication Nos. US2011/0274685 and US 2012/0213771, incorporated by reference herein intheir entireties) anti-ICOS mAbs (for example, MEDI-570, JTX-2011, andanti-TIM3 antibodies (see, for example, WO 2013/006490 or U.S. PatentPublication No US 2016/0257758, incorporated by reference herein intheir entireties), as well as monoclonal antibodies to prostate cancer,ovarian cancer, breast cancer, endometrial cancer, multiple myeloma,melanoma, lymphomas, lung cancers including small cell lung cancer,kidney cancer, colorectal cancer, pancreatic cancer, gastric cancer,brain cancer, (see generally www.clinicaltrials.gov).

4. PVRIG and TIGIT Combination Therapy

There are specific combinations of anti-TIGIT and anti-PVRIG antibodiesthat find use in particular embodiments.

In one embodiment, a biopsy is taken from a tumor from a patient withcancer, and dissociated as is known in the art for FACS analysis. Thecells are stained with labeled antibodies to (1) PVRIG (generally usingCHA.7.518.1H4(S241P), for example, although any outlined inWO2016/134333 (specifically including any that bind, even if they don'tblock) or WO2017/041004) can be used); (2) TIGIT (for example using anydescribed herein or others in the art such as MBSA43); (3) PVR (forexample using those known in the art such as SKII.4) and (4) PVRL2 (forexample using those known in the art such as TX11); and (5) an isotypecontrol antibody. FACS is done, and for each receptor, the percentage ofthe cells expressing the receptor relative to the control antibody iscalculated. If the percentage of positive cells for PVRIG, TIGIT, PVRand PVRL2 is ≥1% for all 4 receptors, then the patient is treated withantibodies to PVRIG and TIGIT. Preferred combinations in this regard areCHA.7.518.1.H4(S241P) and CPA.9.086.

In one embodiment, antibodies containing the CDR sets from theanti-TIGIT antibody CPA.9.086 are combined with antibodies containingthe CDR sets from the anti-PVRIG antibody CHA.7.518.1. In a particularembodiment, antibodies containing the VH and VL sequences from theanti-TIGIT antibody CPA.9.086 are combined with antibodies containingthe VL and VL from the anti-PVRIG antibody CHA.7.518.1. In oneembodiment, CPA.9.086.H4(S241P) as shown in FIG. 53 is combined withCHA.7.518.1H4(S241P) as shown in FIG. 3.

In one embodiment, antibodies containing the CDR sets from theanti-TIGIT antibody CPA.9.083 are combined with antibodies containingthe CDR sets from the anti-PVRIG antibody CHA.7.518.1. In a particularembodiment, antibodies containing the VH and VL sequences from theanti-TIGIT antibody CPA.9.083 are combined with antibodies containingthe VL and VL from the anti-PVRIG antibody CHA.7.518.1. In oneembodiment, CPA.9.086.H4(S241P) is combined with CHA.7.518.1H4(S241P).

In one embodiment, antibodies containing the CDR sets from theanti-TIGIT antibody CPA.9.086 are combined with antibodies containingthe CDR sets from the anti-PVRIG antibody CHA.7.538.1.2.H4(S241P). In aparticular embodiment, antibodies containing the VH and VL sequencesfrom the anti-TIGIT antibody CPA.9.086 are combined with antibodiescontaining the VL and VL from the anti-PVRIG antibodyCHA.7.538.1.2.H4(S241P). In one embodiment, CPA.9.086.H4(S241P) iscombined with CHA.7.538.1.2.H4(S241P).

In one embodiment, CHA.518.1.H4(S241P) is combined with an anti-TIGITantibody as recited in the sequence listing (with reference to all theantibodies listed in FIG. 4 of U.S. Ser. No. 62/513,916), specificallyCPA.9.018, CPA.9.027, CPA.9.049, CPA.9.057, CPA.9.059, CPA.9.083,CPA.9.086, CPA.9.089, CPA.9.093, CPA.9.101, CPA.9.103, CHA.9.536.1,CHA.9.536.3, CHA.9.536.4, CHA.9.536.5, CHA.9.536.6, CHA.9.536.7,CHA.9.536.8, CHA.9.560.1, CHA.9.560.3, CHA.9.560.4, CHA.9.560.5,CHA.9.560.6, CHA.9.560.7, CHA.9.560.8, CHA.9.546.1, CHA.9.546.1,CHA.9.547.2, CHA.9.547.3, CHA.9.547.4, CHA.9.547.6, CHA.9.547.7,CHA.9.547.8, CHA.9.547.9, CHA.9.547.13, CHA.9.541.1, CHA.9.541.3.CHA.9.541.4. CHA.9.541.5, CHA.9.541.6. CHA.9.541.7 and CHA.9.541.8

In one embodiment, CPA.9.086 is combined with an anti-PVRIG antibody asoutlined WO2017/041004, including, but not limited to, those having a) aHC sequence SEQ ID NO:5 and LC sequence SEQ ID NO:3 (or the CDR setscontained therein) b) a HC sequence SEQ ID NO:32 and LC sequence SEQ IDNO:33 (or the CDR sets contained therein); and c) a HC sequence SEQ IDNO:32 and LC sequence SEQ ID NO:40 (or the CDR sets contained therein).

In some embodiments, the combination comprises an anti-TIGIT antibodyselected from the group consisting of CPA.9.086, CPA.9.083, CHA.9.547.7,and CHA.9.547.13 and the PVRIG antibody is selected from the groupconsisting of CHA7.518.1 and CHA.7.538.1.2. In some embodiments, thecombination comprises an anti-TIGIT antibody selected from the groupconsisting of CPA.9.086, CPA.9.083, CHA.9.547.7, and CHA.9.547.13 andthe PVRIG antibody is CHA7.518.1. In some embodiments, the combinationcomprises an anti-TIGIT antibody selected from the group consisting ofCPA.9.086, CPA.9.083, CHA.9.547.7, and CHA.9.547.13 and the PVRIGantibody is CHA7.538.1.2. In some embodiments, the combination comprisesthe anti-TIGIT antibody CPA.9.086 and the PVRIG antibody CHA7.518.1. Insome embodiments, the combination comprises the anti-TIGIT antibodyCPA.9.083 and the PVRIG antibody CHA7.518.1. In some embodiments, thecombination comprises the anti-TIGIT antibody CHA.9.547.7 and the PVRIGantibody CHA7.518. In some embodiments, the combination comprises theanti-TIGIT antibody CHA.9.547.13 and the PVRIG antibody CHA7.518.1. Insome embodiments, the combination comprises the anti-TIGIT antibodyCPA.9.086 and the PVRIG antibody CHA7.538.1.2. In some embodiments, thecombination comprises the anti-TIGIT antibody CPA.9.083 and the PVRIGantibody CHA7.538.1.2. In some embodiments, the combination comprisesthe anti-TIGIT antibody CHA.9.547.7 and the PVRIG antibody CHA7.538.1.2.In some embodiments, the combination comprises the anti-TIGIT antibodyCHA.9.547.13 and the PVRIG antibody CHA7.538.1.2.

FIGS. 20-24 provides PVRIG antibodies, as disclosed in U.S. patentapplication Ser. No. 15/277,978, filed Sep. 27, 2016. The TIGITantibodies of the present invention can be used in combination with thePVRIG antibodies as disclosed in these figures, as well as thosedisclosed throughout this application.

5. Assessment of Treatment

Generally, the antibodies of the invention, alone or in combination(PVRIG with PD-1, TIGIT with PD-1 or TIGIT with PVRIG) are administeredto patients with cancer, and efficacy is assessed, in a number of waysas described herein. Thus, while standard assays of efficacy can be run,such as cancer load, size of tumor, evaluation of presence or extent ofmetastasis, etc., immuno-oncology treatments can be assessed on thebasis of immune status evaluations as well. This can be done in a numberof ways, including both in vitro and in vivo assays. For example,evaluation of changes in immune status (e.g. presence of ICOS+ CD4+ Tcells following ipi treatment) along with “old fashioned” measurementssuch as tumor burden, size, invasiveness, LN involvement, metastasis,etc. can be done. Thus, any or all of the following can be evaluated:the inhibitory effects of PVRIG or TIGIT on CD4⁺ T cell activation orproliferation, CD8⁺ T (CTL) cell activation or proliferation, CD8⁺ Tcell-mediated cytotoxic activity and/or CTL mediated cell depletion, NKcell activity and NK mediated cell depletion, the potentiating effectsof PVRIG or TIGIT on Treg cell differentiation and proliferation andTreg- or myeloid derived suppressor cell (MDSC)-mediatedimmunosuppression or immune tolerance, and/or the effects of PVRIG orTIGIT on proinflammatory cytokine production by immune cells, e.g.,IL-2, IFN-γ or TNF-α production by T or other immune cells.

In some embodiments, assessment of treatment is done by evaluatingimmune cell proliferation, using for example, CFSE dilution method, Ki67intracellular staining of immune effector cells, and 3H-Thymidineincorporation method.

In some embodiments, assessment of treatment is done by evaluating theincrease in gene expression or increased protein levels ofactivation-associated markers, including one or more of: CD25, CD69,CD137, ICOS, PD1, GITR, OX40, and cell degranulation measured by surfaceexpression of CD107A.

In some embodiments, the assessment of treatment is done by assessingthe amount of T cell proliferation in the absence of treatment, forexample prior to administration of the antibodies of the invention. If,after administration, the patient has an increase in T cellproliferation, e.g. a subset of the patient's T cells are proliferating,this is an indication that the T cells were activated.

Similarly, assessment of treatment with the antibodies of the inventioncan be done by measuring the patient's IFNγ levels prior toadministration and post-administration to assess efficacy of treatment.This may be done within hours or days.

In general, gene expression assays are done as is known in the art. Seefor example Goodkind et al., Computers and Chem. Eng. 29(3):589 (2005),Han et al., Bioinform. Biol. Insights 11/15/15 9(Suppl. 1):29-46, Campoet al., Nod. Pathol. 2013 Jan.; 26 suppl. 1:S97-S110, the geneexpression measurement techniques of which are expressly incorporated byreference herein.

In general, protein expression measurements are also similarly done asis known in the art, see for example, Wang et al., Recent Advances inCapillary Electrophoresis-Based Proteomic Techniques for BiomarkerDiscovery, Methods. Mol. Biol. 2013:984:1-12; Taylor et al, BioMed Res.Volume 2014, Article ID 361590, 8 pages, Becerk et al., Mutat. Res 2011Jun. 17:722(2): 171-182, the measurement techniques of which areexpressly incorporated herein by reference.

In some embodiments, assessment of treatment is done by assessingcytotoxic activity measured by target cell viability detection viaestimating numerous cell parameters such as enzyme activity (includingprotease activity), cell membrane permeability, cell adherence, ATPproduction, co-enzyme production, and nucleotide uptake activity.Specific examples of these assays include, but are not limited to,Trypan Blue or PI staining, ⁵¹Cr or ³⁵S release method, LDH activity,MTT and/or WST assays, Calcein-AM assay, Luminescent based assay, andothers.

In some embodiments, assessment of treatment is done by assessing T cellactivity measured by cytokine production, measure either intracellularlyin culture supernatant using cytokines including, but not limited to,IFNγ, TNFα, GM-CSF, IL2, IL6, IL4, IL5, IL10, IL13 using well knowntechniques.

Accordingly, assessment of treatment can be done using assays thatevaluate one or more of the following: (i) increases in immune response,(ii) increases in activation of αβ and/or γδ T cells, (iii) increases incytotoxic T cell activity, (iv) increases in NK and/or NKT cellactivity, (v) alleviation of αβ and/or γδ T-cell suppression, (vi)increases in pro-inflammatory cytokine secretion, (vii) increases inIL-2 secretion; (viii) increases in interferon-γ production, (ix)increases in Th1 response, (x) decreases in Th2 response, (xi) decreasesor eliminates cell number and/or activity of at least one of regulatoryT cells (Tregs).

Assays to Measure Efficacy

In some embodiments, T cell activation is assessed using a MixedLymphocyte Reaction (MLR) assay as is described in the Examples. Anincrease in activity indicates immunostimulatory activity. Appropriateincreases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in immune response as measured for an example byphosphorylation or de-phosphorylation of different factors, or bymeasuring other post translational modifications. An increase inactivity indicates immunostimulatory activity. Appropriate increases inactivity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in activation of αβ and/or γδ T cells as measured for anexample by cytokine secretion or by proliferation or by changes inexpression of activation markers like for an example CD137, CD107a, PD1,etc. An increase in activity indicates immunostimulatory activity.Appropriate increases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in cytotoxic T cell activity as measured for an example bydirect killing of target cells like for an example cancer cells or bycytokine secretion or by proliferation or by changes in expression ofactivation markers like for an example CD137, CD107a, PD1, etc. Anincrease in activity indicates immunostimulatory activity. Appropriateincreases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in NK and/or NKT cell activity as measured for an example bydirect killing of target cells like for an example cancer cells or bycytokine secretion or by changes in expression of activation markerslike for an example CD107a, etc. An increase in activity indicatesimmunostimulatory activity. Appropriate increases in activity areoutlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in αβ and/or γδ T-cell suppression, as measured for an exampleby cytokine secretion or by proliferation or by changes in expression ofactivation markers like for an example CD137, CD107a, PD1, etc. Anincrease in activity indicates immunostimulatory activity. Appropriateincreases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in pro-inflammatory cytokine secretion as measured for exampleby ELISA or by Luminex or by Multiplex bead based methods or byintracellular staining and FACS analysis or by Alispot etc. An increasein activity indicates immunostimulatory activity. Appropriate increasesin activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in IL-2 secretion as measured for example by ELISA or byLuminex or by Multiplex bead based methods or by intracellular stainingand FACS analysis or by Alispot etc. An increase in activity indicatesimmunostimulatory activity. Appropriate increases in activity areoutlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in interferon-γ production as measured for example by ELISA orby Luminex or by Multiplex bead based methods or by intracellularstaining and FACS analysis or by Alispot etc. An increase in activityindicates immunostimulatory activity. Appropriate increases in activityare outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in Th1 response as measured for an example by cytokinesecretion or by changes in expression of activation markers. An increasein activity indicates immunostimulatory activity. Appropriate increasesin activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in Th2 response as measured for an example by cytokinesecretion or by changes in expression of activation markers. An increasein activity indicates immunostimulatory activity. Appropriate increasesin activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases cell number and/or activity of at least one of regulatory Tcells (Tregs), as measured for example by flow cytometry or by IHC. Adecrease in response indicates immunostimulatory activity. Appropriatedecreases are the same as for increases, outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in M2 macrophages cell numbers, as measured for example byflow cytometry or by IHC. A decrease in response indicatesimmunostimulatory activity. Appropriate decreases are the same as forincreases, outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in M2 macrophage pro-tumorigenic activity, as measured for anexample by cytokine secretion or by changes in expression of activationmarkers. A decrease in response indicates immunostimulatory activity.Appropriate decreases are the same as for increases, outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in N2 neutrophils increase, as measured for example by flowcytometry or by IHC. A decrease in response indicates immunostimulatoryactivity. Appropriate decreases are the same as for increases, outlinedbelow.

In one embodiment, the signaling pathway assay measures increases ordecreases in N2 neutrophils pro-tumorigenic activity, as measured for anexample by cytokine secretion or by changes in expression of activationmarkers. A decrease in response indicates immunostimulatory activity.Appropriate decreases are the same as for increases, outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in inhibition of T cell activation, as measured for an exampleby cytokine secretion or by proliferation or by changes in expression ofactivation markers like for an example CD137, CD107a, PD1, etc. Anincrease in activity indicates immunostimulatory activity. Appropriateincreases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in inhibition of CTL activation as measured for an example bydirect killing of target cells like for an example cancer cells or bycytokine secretion or by proliferation or by changes in expression ofactivation markers like for an example CD137, CD107a, PD1, etc. Anincrease in activity indicates immunostimulatory activity. Appropriateincreases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in αβ and/or γδ T cell exhaustion as measured for an exampleby changes in expression of activation markers. A decrease in responseindicates immunostimulatory activity. Appropriate decreases are the sameas for increases, outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases αβ and/or γδ T cell response as measured for an example bycytokine secretion or by proliferation or by changes in expression ofactivation markers like for an example CD137, CD107a, PD1, etc. Anincrease in activity indicates immunostimulatory activity. Appropriateincreases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in stimulation of antigen-specific memory responses asmeasured for an example by cytokine secretion or by proliferation or bychanges in expression of activation markers like for an example CD45RA,CCR7 etc. An increase in activity indicates immunostimulatory activity.Appropriate increases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in apoptosis or lysis of cancer cells as measured for anexample by cytotoxicity assays such as for an example MTT, Cr release,Calcine AM, or by flow cytometry based assays like for an example CFSEdilution or propidium iodide staining etc. An increase in activityindicates immunostimulatory activity. Appropriate increases in activityare outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in stimulation of cytotoxic or cytostatic effect on cancercells. as measured for an example by cytotoxicity assays such as for anexample MTT, Cr release, Calcine AM, or by flow cytometry based assayslike for an example CFSE dilution or propidium iodide staining etc. Anincrease in activity indicates immunostimulatory activity. Appropriateincreases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases direct killing of cancer cells as measured for an example bycytotoxicity assays such as for an example MTT, Cr release, Calcine AM,or by flow cytometry based assays like for an example CFSE dilution orpropidium iodide staining etc. An increase in activity indicatesimmunostimulatory activity. Appropriate increases in activity areoutlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases Th17 activity as measured for an example by cytokine secretionor by proliferation or by changes in expression of activation markers.An increase in activity indicates immunostimulatory activity.Appropriate increases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in induction of complement dependent cytotoxicity and/orantibody dependent cell-mediated cytotoxicity, as measured for anexample by cytotoxicity assays such as for an example MTT, Cr release,Calcine AM, or by flow cytometry based assays like for an example CFSEdilution or propidium iodide staining etc. An increase in activityindicates immunostimulatory activity. Appropriate increases in activityare outlined below.

In one embodiment, T cell activation is measured for an example bydirect killing of target cells like for an example cancer cells or bycytokine secretion or by proliferation or by changes in expression ofactivation markers like for an example CD137, CD107a, PD1, etc. ForT-cells, increases in proliferation, cell surface markers of activation(e.g. CD25, CD69, CD137, PD1), cytotoxicity (ability to kill targetcells), and cytokine production (e.g. IL-2, IL-4, IL-6, IFNγ, TNF-a,IL-10, IL-17A) would be indicative of immune modulation that would beconsistent with enhanced killing of cancer cells.

In one embodiment, NK cell activation is measured for example by directkilling of target cells like for an example cancer cells or by cytokinesecretion or by changes in expression of activation markers like for anexample CD107a, etc. For NK cells, increases in proliferation,cytotoxicity (ability to kill target cells and increases CD107a,granzyme, and perforin expression), cytokine production (e.g. IFNγ andTNF), and cell surface receptor expression (e.g. CD25) would beindicative of immune modulation that would be consistent with enhancedkilling of cancer cells.

In one embodiment, γδ T cell activation is measured for example bycytokine secretion or by proliferation or by changes in expression ofactivation markers.

In one embodiment, Th1 cell activation is measured for example bycytokine secretion or by changes in expression of activation markers.

Appropriate increases in activity or response (or decreases, asappropriate as outlined above), are increases of at least about 5%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 98 to 99% percent overthe signal in either a reference sample or in control samples, forexample test samples that do not contain an anti-PVRIG antibody of theinvention. Specific increases in activity are depicted in FIGS. 27 to34. For example, with regard to increases in T cell proliferation,CHA.7.518.1.H4(S241P) shows an increase of about 60% andCHA.7.538.1.2.H4(S241P) shows an increase of 47%; relevant increases areshown in either T cell proliferation or IFN-γ of from about 10 to 70%with from about 20 to 60% also finding use.

Similarly, increases of at least one-, two-, three-, four- or five-foldas compared to reference or control samples show efficacy.

XII. EXAMPLES

Reference is made to PCT/US2016/18809, filed Feb. 19, 2016, entitled“PVRIG ANTIBODIES AND METHODS OF TREATMENT”, expressly incorporatedherein by reference in its entirety, and in particular for theincorporation of Examples 1-5, 7-8, 11-13, 16-20 and 26-28, and theaccompanying figures.

A. Example 1: Surface Plasmon Resonance Studies of PVR, PVRL2, AND PVRL3Binding to PVRIG, DNAM, and TIGIT

Materials and Methods

All experiments were performed using a ProteOn XPR 36 instrument at 22°C.

Step 1:

A high density goat anti-human fc polyclonal antibody surface(Invitrogen H10500) was prepared over all six lanes of a GLC chip usinga ProteOn XPR 36 biosensor. The activation step for the anti-human fcsurface occurred in the horizontal flow direction while theimmobilization step for the high density pAb occurred in the verticalflow direction. The blocking step occurred in both the vertical andhorizontal positions so that the horizontal “interspots” could be usedas reference surfaces. An average of 4400 RU of goat anti-human pAb wasimmobilized on each lane.

Step 2:

For each cycle, three different lots of human PVRIG fusion protein(human fc, GenScript lots 451, 448, 125), human DNAM-1 fusion protein(human fc, R&D Systems), human TIGIT fusion protein (human fc, R&DSystems), and a control human IgG (Synagis) were each captured over adifferent vertical lane for two minutes at a concentration of 2 μg/mL.PVR, two lots of PVRL2, and PVRL3 were each injected in the horizontalflow direction at six different concentrations over all six capturedligands at different ligand capture cycles. The injections were twominutes followed by 10 minutes of dissociation at a flow rate of504/min. The PVR concentration range was 1.4 nM-332 nM in a 3-folddilution series, both lots of PVRL2 were injected at a concentrationrange of 1.3 nM-322 nM in a 3-fold dilution series, and PVRL3 wasinjected at a concentration range of 1.4 nM-334 nM in a 3-fold dilutionseries. All protein reagents were prepared in running buffer which wasdegassed PBS buffer with 0.05% Tween 20 and 0.01% BSA added. Theanti-human fc capture surfaces were regenerated with two 30-secondpulses of 146 mM phosphoric acid after each cycle.

Step 3:

Sensorgram data of the analytes binding to each captured ligand wereprocessed and double-referenced using ProteOn Manager version 3.1.0.6making use of interspot referencing and a pre-blank injection identicalto the analyte injections.

Results

a) PVR: Binds weakly to captured DNAM-1 and TIGIT and shows no bindingto all three lots of PVRIG and the control IgG. Not enough informationwas generated to estimate the KD of the PVR interactions with DNAM-1 andTIGIT (data not shown).

b) PVRL2: Both lots of PVRL2 showed binding to all three lots of PVRIGand to DNAM-1 but minimal or no binding to TIGIT and no binding to thecontrol IgG. Sensorgrams showed complex kinetics, therefore bindingconstants could not be estimated (data not shown).

c) PVRL3: Showed minimal binding to TIGIT and did not bind the otherproteins (data not shown).

B. Example 2: Effect of PVRIG Knock Down (KD) and Anti-PVRIG Antibody onHuman Melanoma Specific TILS Function

The aim of these assays is to evaluate the functional capacity of PVRIGin human derived TILs, as measured by activation markers and cytokinesecretion, upon co-culture with melanoma target cells.

1. Example 2(1)

The effect of anti-PVRIG antibody (CPA.7.021), which has been shown toblock the interaction of PVRIG and PVRL2, alone or in combination withother antibodies (e.g anti-TIGIT, Anti-DNAM1) was evaluated. PD1 wasused as a benchmark immune-checkpoint for the knock down (siRNA)studies.

Materials and Methods: TILs: Tumor-infiltrating lymphocytes (TILs) fromthree melanoma patients were used (1) TIL-412-HLA-A2-Mart1 specific, (2)TIL-F4-HLA-A2-gp100 specific, and (3) TIL-209-HLA-A2-gp100 specific.TILs were thawed in IMDM (BI, 01-058-1A) full medium supplemented with10% human serum (Sigma, H3667)+1% Glutamax (Life technologies,35050-038)+1% Na-Pyruvate (Biological Industries, 03-042-1B)+1%non-essential amino acids (Biological Industries, 01-340-1B)+1%Pen-Strep (Biological Industries, 03-031-1B)+300 U/ml of rhIL2(Biolegend, 509129).

Tumor cell lines: Human melanoma cells Mel-624 express MART-1 and gp-100antigens in the context of MHC-I haplotype HLA-A2. Cells were culturedin complete DMEM medium supplemented with 10%, 25 mM HEPES buffer, 1%,and 1% Pen-Strep.

Knock down in TILs: Knock-down (KD) of human PVRIG and human PD1 in TILswas done using 100 pmol of Dharmacon ON-TARGETplus human PVRIGsiRNA—SMARTpool (L-032703-02) or Human PD1 siRNA—SMARTpool (L-004435) ornon-targeting siRNA (D-001810-01-05). siRNA were electroporated to TILs(AMAXA, program X-005). Electroporation was done on resting TILscultured in full IMDM supplemented with IL-2 24 hr post thawing. Afterthe electroporation TILs were seeded in 96 well TC plate to recover for24 hr. After 24 hr, cells were harvested and stained with viability dye(BD Horizon; Cat #562247, BD biosciences), washed with PBS and stainedwith anti-human PVRIG—CPA.7.021 (CPA.7.021 IgG2 A647, 7.5 ug/ml) or withanti-human PD-1 (Biolegend, #329910 AF647, 5 ug/ml) in room temperaturefor 30 min. isotype control used are synagis (IgG2 A647, 7.5 ug/ml) andmouse IgG1 (Biolegend #400130 A647, 5 ug/ml) respectively. All sampleswere run on a MACSQuant analyzer (Miltenyi) and data was analyzed usingFlowJo software (v10.0.8).

Co-culture of TILs with 624 melanoma cells: siRNA electroporated TILswere harvested and seeded in 96 TC plate 5×104/well. Mel-624 cells wereharvested as well and seeded in 1:1/1:3 E:T ratios in co-culture. Theplate was incubated overnight (18 hr) in 37° C., 5% CO2.

To assess the effect of anti-PVRIG antibody (CPA.7.021), anti-TIGIT(Clone 10A7; from Genentech US Patent Application No. US 2009/0258013)and anti-DNAM1 (clone DX11, first described in Shibuya et al ImmunityVolume 4, Issue 6, 1 Jun. 1996, Pages 573-581; BD Biosciences; Mouseanti-human DNAM-1 Clone DX11, Cat No. 559787) on melanoma specific TILactivity, TILs (1×105 cells/well) were pre-incubated with testedantibodies or relevant isotype controls in mono-treatment (10 μg/mL) orin combination-treatment (final 10 μg/mL for each) prior to the additionof 624 Melanoma target cells at a 1:1 Effector:Target ratio. The platewas incubated overnight (18 hr) in 37° C., 5% CO2.

Assessment of TILs activation: 16 hours post co-culture, cells werestained with viability dye (BD Horizon; Cat #562247, BD biosciences),washed with PBS and exposed to Fc blocking solution (cat #309804,Biolegend), followed by surface staining with anti-CD8a (Cat #301048,Biolegend) and anti-CD137 (Cat #309804, Biolegend) in 4° C. for 30 min.All samples were run on a MACSQuant analyzer (Miltenyi) and data wasanalyzed using FlowJo software (v10.0.8). Culture supernatants werecollected and analyzed for cytokine secretion by CBA kit (Cat #560484,BD).

Results

PVRIG Knock-Down in TILs:

TIL MART-1 and TIL F4 were cultured 24 hr with IL-2. 100 pmol ofON-TARGETplus human PVRIG siRNA—SMART pool (L-032703-02) or Human PD1siRNA—SMARTpool (L-004435) or non-targeting siRNA (D-001810-01-05) wereelectroporated to TILs (AMAXA, program X-005). Detection of PVRIG orPD-1 was performed 24 hr post electroporation (and prior to co-culture).Cells were stained for viability dye followed by 30 min RT incubationwith anti PVRIG or anti PD-1. The percentage of KD population isindicated in FIG. 82 of U.S. Ser. No. 15/048,967, incorporated byreference herein.

Functional assay using knocked down TILs: Human TILs, cultured for 24hours with IL2 were electroporated with siRNA encoding for human PVRIGor PD-1 or scrambled sequence as control. TILs were tested for PVRIG andPD-1 expression 24 hr post electroporation. ˜80% knock down of PVRIG and˜50% knock down of PD-1 compared to scrambled-electroporated TILs wasobserved as demonstrated in FIG. 82 of U.S. Ser. No. 15/048,967,incorporated by reference herein.

KD TILs were cultured with Mel-624 cells in 1:1 or 1:3 E:T for 18 hr andwere stained for the expression of CD137. Elevated levels of activationmarker CD137 were shown in TIL MART-1 electroporated with PVRIG siRNA,similarly to TILs that were electroporated with PD-1 siRNA, compared tocontrol scrambled siRNA (as demonstrated in FIG. 83A of U.S. Ser. No.15/048,967, incorporated by reference herein). Co-culture supernatantwas collected and tested for the presence of secreted cytokines. TILsthat were electroporated with PVRIG siRNA show a significant increase inIFNγ and TNF levels compared to control SCR siRNA. A similar effect wasshown in TILs that were electroporated with PD-1 siRNA (as demonstratedin FIG. 83B-C of U.S. Ser. No. 15/048,967, incorporated by referenceherein).

The same trend of increase in activation levels was observed in TIL F4.Co-culture of PVRIG siRNA electroporated TIL F4 with Mel-624 in 1:3 E:Tled to increased levels of CD137 surface expression as well as increasedsecretion of IFNγ in co-culture supernatant as shown in FIGS. 84A and84B of U.S. Ser. No. 15/048,967, incorporated by reference herein.Similar trends were observed in TILs that were electroporated with PD-1siRNA.

Functional Assay Using Blocking Abs:

In vitro monotherapy and combo therapy of anti-PVRIG and anti-TIGIT: 209TILs were cultured with Mel-624 cells in 1:1 E:T for 18 hr. Co-culturesupernatant was collected and tested for the presence of secretedcytokines. Treatment with anti TIGIT did not affect IFNγ or TNFsecretion levels. However, an increase in IFNγ and TNF levels wasobserved when anti TIGIT and anti PVRIG were added to co-culture incombination (FIG. 8A-B).

In vitro monotherapy and combo therapy of anti-PVRIG and anti-TIGIT: 209TILs were cultured with Mel-624 cells in 1:1 E:T for 18 hr. TILs werestained for surface expression of activation marker CD137 and showedreduced level of expression upon treatment with anti DNAM-1. Co-culturesupernatant was collected and tested for presence of secreted cytokines.Treatment of anti DNAM-1 mediated a trend to increase secreted cytokinesIFNγ and TNF. Treatment with anti DNAM-1 and anti PVRIG in combinationpartially reversed the effect on CD137 expression (FIG. 9C) and enhancedthe effect on cytokine secretion IFNγ and TNF (FIG. 9A-B). MART-1 TILswere cultured with Mel-624 cells in 1:1 E:T for 18 hr. Co-culturesupernatant was collected and tested for the presence of secretedcytokines. Treatment with anti DNAM-1 reduced CD137 surface expressionon TILs and also the secreted cytokines IFNγ and TNF. Treatment withanti DNAM-1 and anti PVRIG in combination partially reversed theseeffects (FIG. 9D-F).

Summary and conclusions: PD1 KD improved TIL activity, as measured byIFNγ and secretion in F4 and MART-1 TILs. An increase (˜20%) of IFNγ andTNF secretion was observed upon PVRIG KD in MART-1 TILs compared tocontrol siRNA. The same trend was observed in CD137 expression uponco-culture with 624 Melanoma cells on F4 TILs.

Treatment of anti-TIGIT did not affect IFNγ or TNF secretion levels fromTILs co-cultured with 624 Mels, however, an increase in IFNγ and TNFlevels was observed when anti TIGIT and anti PVRIG (CPA.7.021) wereadded to co-culture in combination.

Anti DNAM-1 treatment reduced TIL-MART-1 activation manifested byreduced CD137 and cytokine secretion and anti-PVRIG (CPA.7.021) couldpartially reverse this effect in combo treatment with DNAM-1 Ab. In TIL209, IFNγ and TNF secretion levels were slightly elevated (˜10%) withanti DNAM-1, and an increase in IFNγ and TNF levels (˜40% and 30%,respectively) was observed when anti DNAM1 and anti PVRIG (CPA.7.021)were added to co-culture in combination. Collectively, our resultsshowed that PVRIG is a new co-inhibitory receptor for PVRL2.

2. Example 2(2)

The effect of additional anti-PVRIG antibodies (CHA.7.518.1.H4(S241P);CHA.7.524; CHA.7.530; CHA.7.538), which have been shown block theinteraction of PVRIG and PVRL2, alone or in combination with otherantibodies (e.g anti-TIGIT, PD1) on TIL-209, TIL-412 and TIL-463-F4activity upon co-culture with 624 melanoma cell line was evaluated.

Functional antibodies used in this assay were anti hPVRIG hybridoma Abs(mIgG1 backbone)—CHA.7.518.1.H4(S241P); CHA.7.524; CHA.7.530; CHA.7.538(M1 lot #30816); anti hTIGIT (mIgG1 backbone)—clone 10A7 (Genescript),anti-TIGIT clone MBSA43 (e-biosciences) and mIgG1 (cat #400166, MOPC-21clone, Biolegend)

Co-culture of TIL and 624 mels: TILs were thawed and cultivated asdescribed in 2.1 24 hr prior to co-culture. Abs tested were added inmono-treatment (10 ug/mL) or in combination with anti TIGIT (20 ug/mL)to seeded TILs and incubated (in total 100 uL) for 1 hr in 37° C., 5%CO2. Mel-624 cells were harvested and seeded in 1:3 Effector: Targetratio in co-culture with TILs. Plate was incubated overnight (18 hr) in37° C., 5% CO2.

Assessment of TILs functional capacity: T cell activity was assessedbased on detection of IFNγ in co-culture supernatants. Culturesupernatants were collected and tested for cytokines by CBA kit (Cat#560484, BD) or by MAGPIX human IFNγ/TNFα kit. Two tailed unpairedT-tests were calculated. P<0.05 was referred to as statisticallysignificant.

Results

Functional Assay Using TILs and Melanoma Cells in the Presence of AntiPVRIG Hybridoma Abs:

Human TILs, cultured for 24 hours with IL2 were co-cultured with Mel-624cells in 1:3 E:T for 18 hr and tested for cytokine secretion. FIG. 31described a representative experiment out of 5-6 performed. TILs wereco-cultured with melanoma cells 624 in the presence of anti-TIGIT oranti-PVRIG Abs (blue) or in combination of anti-TIGIT and anti PVRIG(green) and tested for IFNγ/TNF secretion. In this experiment, all 4anti-PVRIG Abs mono treatments increased (20-30%) IFNγ secretion in 2out of 3 TILs tested (TIL-209 and TIL463-F4) while in combination withanti-TIGIT all anti-PVRIG Abs CHA.7.518.1.H4(S241P), CHA.7.530,CHA.7.538 increased IFNγ secretion compared to anti-TIGIT treatmentalone.

The effect of Ab CHA.7.518.1.H4(S241P) was found statisticallysignificant across experiments in TIL 463-F4-gp100 across 5 experimentsas mono and in combination with anti-TIGIT (FIG. 9E, G). Combo treatmentof anti-PVRIG Ab CHA.7.518.1.H4(S241P) effect was also statisticallysignificant in TIL 209 (FIG. 9C). Combo treatment effect of anti PVRIGAb CHA.7.538 was found statistically significant in TIL 463-F4-gp100(FIG. 9F).

Summary and Conclusions:

In the experimental systems described herein we observed an effect ofanti PVRIG on TILs in response to target melanoma cells as seen bychanges in IFNγ secretion. Anti PVRIG Hybridoma Abs tested mediated anincrease in IFNγ secretion compared to relevant isotype control. AbCHA.7.518.1.H4(S241P) seems to have an advantage in mediating anincrease in IFNγ secretion as a mono-treatment and compared to otheraPVRIG Abs tested however the magnitude of this effects varies betweendifferent TILs. This effect is enhanced in combination with anti-TIGITtreatment.

3. Example 2(3)

The aim is to evaluate the functional activity of anti-human PVRIGantibodies (CHA.7.518.1.H4(S241P); CHA.7.544; or CHA.7.538) on humanTILs activity upon co-culture with peptide-pulsed CHO-S cells stablyco-expressing HLA-A2, b2 microglobulin (B2M) and PVRL2.

TILs from resected metastases of three melanoma patients were used:TIL-412-HLA-A2-Mart1(26-35) specific, TIL-463-F4-HLA-A2-gp100(209-217)specific, TIL-463-F5-HLA-A2-gp100(209-217) specific, andTIL-209-HLA-A2-gp100(209-217) specific.

TILs were thawed in IMDM full medium supplemented with 10% humanserum+1% Glutamax+1% Na-Pyruvate+1% non-essential amino acids+1%Pen-Strep+300 U/ml of rhIL2 (Biolegend, 589106).

CHO-S cells (target cells) were stably transduced with a lentivirusexpressing HLA-A2/B2M (lentivirus vector cat # CD515B-1-SBI, systembiosciences) and grown under 600 ug/ml of hygromycin B selection in CDCHO medium (Cat #10743-011) supplemented with 8 mM GlutaMax 1% and 1%Pen/Strep. HLA-A2/B2M expressing cells were then cloned by limitingdilution. The 3E8 clone with high HLA-A2 and B2M expression was thentransduced with a lentivirus expressing human PVRL2 (lentivirus vectorcat # CD510B-1-SBI, system biosciences), and grown under 6 ug/mlpuromycin selection.

In the experimental system described herein (depicted in FIG. 35), gp100or MART-1-reactive TILs that endogenously express TIGIT, DNAM-1 andPVRIG FIG. 37) were co-cultured with peptide-pulsed CHO-SHLA-A2/B2M/PVRL2 cells.

Functional antibodies used in this assay were anti human PVRIG; Ab 461(Aldeveron)—referred to 544 in this example, anti human PVRIG chimera Ab(hIgG4 back bone)—CHA.7.538; CHA.7.518 (referred to c538 and c518 inthis example, meaning that the variable heavy and light regions from7.538 and 7.518 were fused to human IgG4 constant regions anti humanTIGIT (mIgG1 backbone) clone MBSA43 (e-biosciences), mIgG1 (biolegend)and hIgG4 (biolegend).

TILs were thawed and cultured as described herein for 24 hr prior toco-culture with target cells. The tested antibodies were added inmono-treatment (10 ug/mL) or in combination with anti TIGIT (total 20ug/mL) to seeded TILs and incubated (in total 100 uL) for 30 min in 37°C., 5% CO2. CHO-S cells were harvested and pulsed with 0.1 or 0.5 ug/mlof gp-100 (gp100209-217) or with 20 ug/ml of MART-126-35 peptides, for 1hour at 37° C. in Opti-MEM™ reduced serum media. Following tree washeswith Opti-MEM™ reduced serum media, peptide-pulsed target cells wereover-night (18 hr) co-cultured with TILs at Effector:Target ratio of 1:3(33 k:100 k).

Assessment of TILs functional capacity: The effect of anti PVRIGantibodies (10 ug/ml) as mono treatment or as combo treatment with antiTIGIT on TILs activity was assessed using measurement of cytokinessecretion from over-night co-culture supernatants using Combined BeadArray (CBA) kit (Cat #560484, BD). All samples were acquired inMACSQuant analyzer (Miltenyi) and data was analyzed using FlowJosoftware (v10.0.8).

Dose response of anti PVRIG antibodies: The effect of anti PVRIGantibodies c518, c538 (or hIgG4 isotype control) dose response wastested on the described assay in an antibodies concentration of 30, 10,3, 1, 0.3, 0.1 and 0.03 ug/ml. Two tailed unpaired T-tests werecalculated. P<0.05 was referred to as statistically significant.

Results

Effect of anti PVRIG antibodies on TILs activity upon co-culture withCHO-S HLA-A2/B2M cells expressing PVRL2: The effect of three anti PVRIGantibodies (544, c538 and c518) on the activity of four different TILs(412, 463, 462 and 209) from two different experiments is summarized inFIG. 37. Ab served as non-blocker Ab control. The detailed results ofthe experiments are presented in FIG. 39. Treatment with 544, c538 andc518 antibodies increased the levels of IFN secretion from TILs (onaverage of 6%, 28% and 23%, respectively) compared to treatment withisotype antibody. Increased IFN secretion was detected in TILs treatedwith c538 or c518 compared to 544, the non-blocker control. Nosignificant difference was found between treatments with c538 to c518Abs. Treatment with anti TIGIT increased IFN secretion from TILs (onaverage of 49%) compared to isotype. The combo treatment of c518 andc538 with anti TIGIT induced additive effect in IFN secretion from TILs,but the combo effect was not statistically significant compare totreatment with mono treatment of TIGIT.

Effect of anti-PVRIG antibodies dose response on TILs functionalcapacity: The effect of adding anti PVRIG antibodies (c538 and c518) indose response on the activity of TILs F4 and 209 was evaluated (FIG.80). The EC50 of c518 and c538 antibodies is in the single digit nMcompared to isotype control as measured by the effect of TNFα secretionfrom the TILs.

Summary and Conclusions:

In the experimental system described herein we observed effect of antiPVRIG antibodies on TILs activity in response to co-culture withpeptide-pulsed CHO-S HLA-A2/B2M target cells over-expressing PVRL2. Theanti PVRIG antibodies that were tested mediated an increased secretionof IFN and TNF from TILs compared to the relevant isotype control.Antibodies c518 and c538 have statistical significant advantage(p-0.0063 and p-0.0034 respectively) on TIL activity, as manifested byIFN secretion, as compared to 544, which is a non-blocker antibody ofPVRIG (based on competition experiment done on PVRIG expressing cells).Both c518 and c538 antibodies had an additive effect with anti TIGITantibody (no statistical significant).

4. Example 2(4)

The aim of this example was to evaluate the functional capacity of PVRIGin human derived TILs as measured by cytokine secretion upon co-culturewith melanoma target cells. The effect of anti-PVRIG antibodies(CHA.7.518.1.H4(S241P); CHA.7.524; CHA.7.530; CHA.7.538), which havebeen shown block the interaction of PVRIG and PVRL2, alone or incombination with other antibodies (e.g anti-TIGIT, PD1) was evaluated.

Purified CD3+ T cells were obtained using Rossetesep human T cellenrichment cocktail kit (Stem cell technologies) on buffy coat bloodsamples. Cells were thawed and labeled with CFSE (Moleculare probes) tobe able to track proliferation in co-culture.

CHO-S-OKT3 cells: CHO-S cells were transduced with CDSL-OKT3-scFv-CD14in CD710B-1 (SBI, cat # CS965A-1, lot #151014-005, 1.40×108 ifus/ml).Cells were cultured in the presence of CD CHO (Gibco, life technologiesCat #10743-011) with addition of 8 mM GlutaMax and 6 μg/ml puromycin.Surface OKT3 levels were evaluated by flow cytometry using PE-goatanti-mouse IgG F(ab)′2 at 1:200 dilution (Jackson Immunoresearch, cat#115-116-146). CHO-S-OKT3 cells were then transiently transfected withhuman PVRL2 (delta isoform) or empty vector using Amaxa electroporationsystem (Lonza, Walkersville, Md., USA) according to the manufacturer'sinstructions. 5 ug of pcDNA3.1 plasmid (empty vector or hPVRL2) per2×106 cells in Ingenio™ Electroporation Solution (Mirus, Cat #MC-MIR-50115) and pulse-program U-024 were used. Expression of PVRL2 ontransfected CHOS—S-OKT3 cells was evaluated by flow cytometry usinganti-PVRL2 Ab (cat #337412, Biolegend).

The functional antibodies used in this assay were Anti hPVRIG hybridomaAbs (mIgG1 backbone)—CHA.7.518.1.H4(S241P); CHA.7.524; CHA.7.530;CHA.7.538, anti-TIGIT clone MBSA43 (e-biosciences) and mIgG1 (cat#400166, MOPC-21 clone, Biolegend).

Co-culture of CD3 T cells and CHO-OKT3 cells: CD3+ T cells were thawedand immediately labeled with CFSE. In parallel CHO-S-OKT3-PVRL2 cellswere harvested and treated with Mitomycin-C for 1 hr in 37° C., washedand added to co-culture with T cells in 1:5 E:T (1×105 T cells and 2×104CHO-OKT3-PVRL2 or mock). Abs were added in mono-treatment (10 ug/mL) orin combination with anti TIGIT (10 ug/mL) and co-culture plates wereincubated 37° C., 5% CO2 for 5 days. After 5 days cells were harvestedand T cell proliferation wad analyzed by FACS gating on CD4 and CD8sub-populations.

Effect of anti-PVRIG antibodies in CHOS-OKT3 co-culture assay:CFSE-labeled T cells were stimulated with stimulator cells (CHO cellsexpressing membrane-bound anti-CD3 mAb fragments). CHOS-stimulator cellsexpressing human PVRL2 and control stimulator cells (empty vector)treated with mitomycin C (50 ug/ml for 1 h) before co-cultured withCFSE-labeled human T cells at the ratio of 1:5. After 5 days at 37° C.and 5.0% CO2, the effect of anti-PVRIG antibodies (10 ug/ml) on T cellproliferation (CFSE dilution) and cytokine secretion (ELISA or TH1/2/17CBA kits) in culture supernatants was assessed. All samples wereacquired in MACSQuant analyzer (Miltenyi) and data was analyzed usingFlowJo software (v10.0.8). Culture supernatants were collected andanalyzed for cytokine secretion by CBA kit (Cat #560484, BD).

Results

Effect of anti-PVRIG antibodies on PVRL2 over-expression in CHOS-OKT3assay: CHOS-OKT3 overexpressing PVRL2 or mock (empty vector) cells wereco-cultured with CD3+ cells and the effect of anti-PVRIG antibodies asmono treatment or in combination with anti-TIGIT on T cell proliferationand cytokine secretion was tested (FIG. 40). After 5 days cells wereharvested and analyzed for CFSE dilution. In parallel co-culturesupernatant was collected and tested for cytokine secretion. FIG. 41shows the effect of anti-PVRIG Abs in responder vs. non responder donor.The effect of various anti-PVRIG Abs on T cell proliferation as monotreatment in combination with anti-TIGIT were evaluated. While someanti-PVRIG Ab enhance T cell proliferation, no additive effect withanti-TIGIT antibody was observed in this system (FIG. 42). These effectswere not seen when the Abs were tested in co-culture of CD3+ cells withmock (empty vector transfected) CHO-S cells (data not shown).

Total of 10 donors were tested and 5 out of 10 donors responded toanti-PVRIG Abs. Treatment of Ab CHA.7.518.1.H4(S241P) consistentlyresulted in enhanced IFNγ secretion ranging between 20-50% across 5responder donors tested while treatment with other Abs did notdemonstrate a clear trend (FIG. 43). Similar effects were observed inCD8+ cells proliferation. Effect of Abs treatment are summarized in FIG.44.

C. Example 3: Effect of Anti-PVRIG Antibody on Human Melanoma SpecificTILS Function in Combination with Anti-TIGIT and Anti-PD1 Antibodies 1.Example 3(1)

Materials and Methods

TILs: Tumor-infiltrating lymphocytes (TILs) from three melanoma patientswere used: (1) TIL-412-HLA-A2-Mart1 specific, (2) TIL-F4-HLA-A2-gp100specific and (3) TIL-209-HLA-A2-gp100 specific.

TILs were thawed in IMDM (BI, 01-058-1A) full medium supplemented with10% human serum (Sigma, H3667)+1% Glutamax (Life technologies,35050-038)+1% Na-Pyruvate (Biological Industries, 03-042-1B)+1%non-essential amino acids (Biological Industries, 01-340-1B)+1%Pen-Strep (Biological Industries, 03-031-1B)+300 U/ml of rhIL2(Biolegend, 509129).

Tumor cell lines: Human melanoma cells Mel-624 express MART-1 and gp-100antigens in the context of MHC-I haplotype HLA-A2. Cells were culturedin complete DMEM medium (Biological Industries, 01-055-1A) supplementedwith 10% FBS (BI, 04-127-1A), 25 mM HEPES buffer (BI, 03-025-1B), 1%Glutamax (Life technologies, 35050-038), and 1% Pen-Strep (BiologicalIndustries, 03-031-1B).

Co-culture of TILs with 624 melanoma cells in the presense ofanti-PVRIG, anti-TIGIT and PD1 blocking antibodies: To assess the effectof anti-PVRIG antibody (CPA.7.021), anti-TIGIT (Clone 10A7) and anti-PD1(mAb 1B8, Merck) on melanoma specific TIL activity, TILs (3×104cells/well) were pre-incubated with tested antibodies or relevantisotype controls in mono-treatment (10 μg/mL) or incombination-treatment (final 10 μg/mL for each) prior to addition of 624Melanoma target cells at 1:3 Effector:target ratio. Plate was incubatedovernight (18 hr) in 37° C., 5% CO2.

Assessment of TILs activation: Culture supernatants were collected andanalyzed for cytokine secretion by CBA kit (Cat #560484, BD).

In vitro monotherapy anti-PVRIG and combo-therapy of with anti-TIGIT andPD1 blocking antibodies: F4 TILs (gp100 sepecific) were cultured withMel-624 cells in 1:3 E:T for 18 hr. Co-culture supernatant was collectedand tested for presence of secreted cytokines. Treatment of anti-TIGITor anti-PD1 did not affect IFNγ or TNF secretion levels. However, anincrease in IFNγ and TNF levels was observed when anti TIGIT or anti-PD1in combination with anti PVRIG were added to co-culture in combination(FIG. 10A-B).

Treatment of anti-PVRIG, anti-TIGIT and PD1 alone did not affect IFNγ orTNF secretion levels from TILs co-culture with 624 Mels, however, anincrease in IFNγ and TNF levels was observed when anti-TIGIT or anti-PD1antibodies were added in combination with anti PVRIG (CPA.7.021). Thepresented data suggest that there is synergestic effect for combinatorytherapy with anti-TIGIT or anti-PD1 antibodies.

2. Example 3(2)

Again, the ability of anti-PVRIG antibodies to enhance CD4+ and CD8+ Tcell function in combination with an anti-TIGIT antibody in a primary invitro cell-based assay was assessed.

CHO-S OKT3 assay: The CHO-S OKT3 assay was utilized to determine whetherthe combination of a humanized PVRIG antibody, CHA.7.518.1.H4(S241P),and a commercially available anti-TIGIT antibody could increase T cellproliferation, and cytokine secretion greater than a single anti-PVRIGor anti-TIGIT antibody treatment. The target cells used in theco-culture assay were the Chinese hamster ovary cell line, CHO-S(ATCC),stably overexpressing the single chain variable fragment of theanti-human CD3 antibody Clone OKT3 (abbreviated as OKT3), and humanPVRL2 (abbreviated as hPVRL2). CHO-S OKT3 parental cells were grown inserum-free CD-CHO medium supplemented with 40 mM glutamax,penicillin/streptomycin, and 6 μg/ml puromycin. CHO-S OKT3 hPVRL2 cellswere grown in serum-free CD-CHO medium supplemented with 40 mM glutamax,penicillin/streptomycin, 6 μg/ml puromycin, and 600 μg/ml hygromycin B.

Primary CD3+ and CD8+ T cells were isolated from healthy human donorsusing the RosetteSep™ human CD3+ T cell enrichment cocktail (StemcellTechnologies), and the human CD8+ microbeads (Miltenyi Biotec),respectively, and frozen in liquid nitrogen. On the day of theco-culture assay, CD3+ or CD8+ T cells were thawed, counted, and labeledwith 1 μM CFSE (Life Technologies) for 10 minutes at 37° C. Followingthis incubation, T cells were washed and resuspended in complete mediumcontaining RPMI, supplemented with 10% heat-inactivated FBS, glutamax,penicillin/streptomycin, non-essential amino acids, sodium pyruvate, and50 μM β-mercaptoethanol. CHO-S OKT3 hPVRL2 cells were harvested fromculture, and treated with mitomycin C for 1 hour at 37° C. with periodicmixing. After the incubation, the target cells were thoroughly washed,counted, and resuspended in complete RPMI medium. The assay was set upwith a 5:1 ratio of T cells (100,000) to target cells (20,000). Thetarget cells, T cells, and 10 ug/ml of each antibody treatment wereadded together in a 96-well U-bottom plate (Costar), and incubated foreither 3 days (CD8+ T cells), or 5 days (CD4+ T cells) at 37° C. Theantibody treatments included human CHA.7.518.1.H4(S241P) IgG4 alone, ahuman IgG4 isotype control combined with the mouse anti-human TIGIT(Clone MBSA43, eBioscience), and a combination of CHA.7.518.1.H4(S241P)and anti-TIGIT (Clone MBSA43). In addition, the activity of the mouseanti-human DNAM-1 IgG1 (Clone DX11, BioLegend), mouse IgG1 isotypecontrol (Clone MOPC21, BioLegend), and a human IgG4 isotype control wasalso assessed.

After the 3 or 5-day incubation period, co-culture supernatants wereanalyzed for secreted cytokines, including IL-2, IL-4, IL-5, IL-6, IL-9,IL-10, IL-13, IL-17A, IL-17F, IL-21, IL-22, TNFα, and IFNγ, with thecytometric bead array (CBA) human Th1/Th2/Th17 cytokine kit (BDBiosciences), or with the LEGENDplex™ Human Th cytokine kit (BioLegend).T cell proliferation was measured by staining CD4+ or CD8+ T cells withthe LIVE/DEAD fixable aqua dead cell stain kit (ThermoFisherScientific), anti-CD4 antibody (Clone RPA-T4, BioLegend), and anti-CD8antibody (Clone HIT8a, BioLegend), and gating on the percentage of live,CFSE low proliferating CD4+ or CD8+ T cells. Data was acquired using aFACS Canto II (BD Biosciences), and analyzed using FlowJo (Treestar) andPrism (Graphpad) software.

Results: Combination of CHA.7.518.1.H4(S241P) and an anti-TIGIT antibodyaugments CD4+ T cell proliferation compared to single antibodytreatments: The ability of CHA.7.518.1.H4(S241P) humanizedhybridoma-derived PVRIG antibody to enhance primary CD4+ T cellproliferation in vitro when combined with an anti-TIGIT antibody wasassessed with the CHO-S OKT3 assay.

FIGS. 33A and B show the percentage of proliferating CD4+ T cells fromtwo different donors in response to co-culture with the CHO-S OKT3hPVRL2 target cells, and treated with anti-PVRIG and anti-TIGITantibodies either alone or in combination. In these two representativehuman CD3+ T cell donors, the combination of CHA.7.518.1.H4(S241P) andthe anti-TIGIT antibody increases CD4+ T cell proliferation compared toCHA.7.518.1.H4(S241P) alone, or the combination of IgG4 isotype and theanti-TIGIT antibody. The anti-DNAM-1 antibody reduces CD4+ T cellproliferation compared to the IgG1 isotype control in both donors.

CHA.7.518.1.H4(S241P) and an anti-TIGIT antibody enhances CD8+ T cellproliferation and IFN-g secretion FIG. 34A illustrates the ability ofthe humanized PVRIG antibody, CHA.7.518.1.H4(S241P), to increase CD8+ Tcell proliferation in combination with the anti-TIGIT antibody in theCHO-S OKT3 assay. In a representative human CD8+ T cell donor, thecombination of CHA.7.518.1.H4(S241P) and the anti-TIGIT antibodyincreases CD8+ T cell proliferation when T cells are co-cultured withthe CHO-S OKT3 hPVRL2 cells. The combination of anti-PVRIG andanti-TIGIT antibodies increases proliferation greater thanCHA.7.518.1.H4(S241P) alone, or the hIgG4 isotype plus anti-TIGITantibody treatment. FIG. 34B shows that in the same representative humanCD8+ T cell donor as described above, the humanized PVRIG antibody,CHA.7.518.1.H4(S241P), in combination with the anti-TIGIT antibody alsoenhances IFNγ secretion in the CHO-S OKT3 assay. The combination ofanti-PVRIG and anti-TIGIT antibodies increases IFNγ secretion greaterthan CHA.7.518.1.H4(S241P) alone, or the hIgG4 isotype plus anti-TIGITantibody treatment. The anti-DNAM-1 antibody reduces both CD8+ T cellproliferation and IFNγ production compared to the IgG1 isotype controlantibody.

Summary and Conclusions

Together, the humanized PVRIG antibody, CHA.7.518.1.H4(S241P) and theanti-TIGIT antibody had in vitro functional activity in the primarycell-based CHO-S OKT3 assay. The combination of CHA.7.518.1.H4(S241P)and the anti-TIGIT antibody led to increased CD4+ and CD8+ T cellproliferation, as well as IFNγ secretion from CD8+ T cells compared totreatment with either CHA.7.518.1.H4(S241P) or the anti-TIGIT antibodyalone. Together, these data demonstrate that co-blockade of the twocheckpoint receptors, PVRIG and TIGIT, increased T cell functioncompared to single receptor blockade.

It should be noted that TIGIT does not interact with CD112 (PVRL2; seeFIGS. 4E and 4F of Zhu et. al., J. Exp. Med. (2016):1-10); rather, itinteracts with PVR, a different ligand. PVR is expressed in theCHO/CD112 system of Zhu et al. Accordingly, our interpretation of thecombination effect of the aCD112R (anti-PVRIG antibody) and anti TIGITis that the aCD112R/aPVRIG is blocking the interaction of human CD112Rwith human CD112, but the anti TIGIT antibody is blocking theinteraction of human TIGIT with human or hamster PVR (on T cells or CHOcells), Zhu et al do not really give a hypothesis as to why the antiCD112R/anti TIGIT combination effect is occurring in the CHO CD112assay. That is, the combination effect is not through the PVRL2/CD112ligand alone.

D. Example 4: Epitope Mapping of Anti-Human PVRIG Antibodies Based onCynomolgus Cross-Reactivity

Rationale and Objectives

The objective of this study is to identify the epitopes on the PVRIGprotein that determine cross-reactivity of anti-human PVRIG antibodiesagainst the cynomolgus monkey (cyno) orthologue. Many of the antibodiesagainst human PVRIG target show varied degrees of cyno cross-reactivitydespite the fact that many of these antibodies belong to the sameepitope bin. To shed light on the molecular basis of human/cynocross-reactivity (or lack thereof), several cyno-to-human mutations ofthe PVRIG recombinant proteins were designed, expressed and purified,and tested for binding to a panel of anti-human PVRIG antibodies inELISA.

Methods

Design of Cyno-to-Human PVRIG Variants:

Sequence alignment of human and PVRIG ECDs shows 90% sequence identityand 93% sequence homology between human and cyno orthologs. Based on thenature of the mutations (conserved vs non-conserved) and the secondarystructure prediction (coil vs extended) of the mutation region, threesite-directed mutants of the cyno PVRIG were designed to probe thecyno-cross reactivity focused epitope mapping. These mutants includeH61R, P67S, and L95R/T97I cyno PVRIG. Wild type cyno and human PVRIGwere also generated.

Expression and purification of cyno, human, and hybrid PVRIG variants:All the PVRIG variants were expressed as ECD fusions with a C-terminal6×His tag in mammalian cells. The proteins were purified by affinitypurification, ion-exchange chromatography, and size-exclusionchromatography. The purified proteins were buffer-exchanged into PBSbuffer (pH 7.4) and stored at 4° C.

ELISA to determine PVRIG-antibody interaction: The functional ELISA wasperformed as follows: cyno, human, and cyno/human hybrid PVRIG(His-tagged) recombinant proteins were adsorbed on an IA plate overnightat 4° C. Coated plate wells were rinsed twice with PBS and incubatedwith 300 μL blocking buffer (5% skim milk powder in PBS pH 7.4) at roomtemperature (RT) for 1 hr. Blocking buffer was removed and plates wererinsed twice more with PBS. Plate-bound PVRIG variants were incubatedwith anti-human PVRIG mAbs (human IgG1 isotype) in solution (linearrange of 0.1 μg/mL to 8 μg/mL in a 50 μL/well volume) at RT for 1 hr.Plates were washed three times with PBS-T (PBS 7.4, 0.05% Tween20), thenthree times with PBS and 504/well of a HRP-conjugated secondary antibodywas added (Human IgG Fc domain specific, Jackson ImmunoResearch). Thiswas incubated at RT for 1 hr and plates were washed again. ELISA signalswere developed in all wells by adding 50 μL of Sureblue TMB substrate(KPL Inc) and incubating for 5-20 mins. The HRP reaction was stopped byadding 50 μL 2N H2SO4 (VWR) and absorbance signals at 450 nm were readon a SpectraMax (Molecular Devices) or EnVision (PerkinElmer)spectrophotometer. The data were exported to Excel (Microsoft) andplotted in GraphPad Prism (GraphPad Software, Inc.).

Results

S67, R95, and 197 Residues as Determinants of Cyno Cross-Reactivity:

The binding data shown in FIG. 18 clearly shows that the S67, R95, and197 residues affect the cyno cross-reactivity of various antibodies.While the P67S cyno-to-human mutation negatively impacts the binding ofCPA.7.002 and CPA.7.041, the L95R/T97I cyno-to-human mutationsignificantly improves the binding of CPA.7.002, CPA.7.021, CPA.7.028,and CPA.7.041. On the other hand, H61R cyno-to-human mutation does notaffect the binding of any of the antibodies tested.

Relative binding to cyno-to-human variants suggests three epitopegroups: The relative binding of the antibodies to cyno, human and hybridPVRIG variants suggests 3 distinct epitope groups: Group 1 binds toR95/I97 residues (CPA.7.021 and CPA.7.028). Group 2 binds to S67 andR95/I97 residues (CPA.7.002 and CPA.7.041). Group 3 does not bind to S67or R95/I97 residues (CPA.7.024 and CPA.7.050). The epitope groups showstrong correlation to the degree of cyno cross-reactivity of theseantibodies (FIG. 19).

Summary and Conclusions:

The restricted epitope mapping based on cyno-to-human variations in thePVRIG ECD identified S67, R95, and 197 residues as determinants of cynocross-reactivity of anti-human PVRIG antibodies. The completerestoration of binding to L95R/T97I cyno PVRIG for CPA.7.021 andCPA.7.028 antibodies and improved binding of CPA.7.002 to this mutantstrongly suggests that R95 and 197 residues are critical human PVRIGepitopes for these antibodies. These findings also suggest a possibleway to predict cross-reactivity to non-human primate PVRIG orthologsbased on their primary amino acid sequence.

E. Example 5: Humanized Antibodies: Binding and Receptor-Ligand BlockingAnalysis of Humanized Anti-PVRIG Hybridoma-Derived Antibodies,CHA.7.518.1.H4(S241P) and CHA.7.538.1.2.H4(S241P)

This experiment was run to characterize the binding ofCHA.7.518.1.H4(S241P) and CHA.7.538.1.2.H4(S241P) to human andcynomolgus PVRIG protein on cell lines and primary leukocytes, tocharacterize the capacity of CHA.7.518.1.H4(S241P) andCHA.7.538.1.2.H4(S241P) to block the interaction between PVRIG and PVRL2and to characterize the epitope space of CHA.7.518.1.H4(S241P) andCHA.7.538.1.2.H4(S241P) relative to each other, by assessing competitionfor binding to PVRIG antigen expressed on Jurkat cells.

FACS analysis of hPVRIG over-expressing cells: The following cell lineswere used to assess the specificity of CHA.7.518.1.H4(S241P) andCHA.7.538.1.2.H4(S241P): HEK parental and HEK hPVRIG over-expressingcells. These cells were cultured in DMEM (Gibco)+10% fetal calf serum(Gibco)+glutamax (Gibco). For the HEK hPVRIG over-expressing cells, 0.5ug/ml puromycin (Gibco) was also added to the media for positiveselection. For FACS analysis, all cell lines were harvested in log phasegrowth and 50,000-100,000 cells per well were seeded in 96 well plates.Binding of unconjugated CHA.7.518.1.H4(S241P) andCHA.7.538.1.2.H4(S241P) (hIgG4) and their respective controls wereassessed in an 8-point titration series starting at 10 ug/ml on ice for30 mins-1 hr. The titration series was conducted as 3 fold serialdilutions. Unconjugated primary antibodies were detected with ananti-human Fc Alexa 647 conjugated antibody (Jackson Laboratories). Datawas acquired using a FACS Canto II (BD Biosciences), FACS LSR FortessaX-20 (BD Biosciences), or IntelliCyt (IntelliCyt Corporation) andanalyzed using FlowJo (Treestar) and Prism (Graphpad) software.

FACS analysis of human cell lines for hPVRIG: The following cell lineswere used to assess the expression and specificity ofCHA.7.518.1.H4(S241P) and CHA.7.538.1.2.H4(S241P): Jurkat and HepG2.Jurkat cells were cultured in RPMI media+10% fetal calf serum, glutamax,non-essential amino acids (Gibco), sodium pyruvate (Gibco), andpenicillin/streptomycin (Gibco). HepG2 cells were cultured in DMEM+10%fetal calf serum+glutamax. For FACS analysis, all cell lines wereharvested in log phase growth and 50,000-100,000 cells per well wereseeded in 96 well plates. Binding of unconjugated CHA.7.518.1.H4(S241P)and CHA.7.538.1.2.H4(S241P) (hIgG4) and their respective controls wereassessed in an 8-point titration series starting at 10 ug/ml on ice for30 mins-1 hr. Unconjugated primary antibodies were detected with ananti-human Fc Alexa 647 conjugated antibody. The titration series wereconducted as 3-fold serial dilutions. Data was acquired using a FACSCanto II or IntelliCyte and analyzed using FlowJo and Prism software.

FACS analysis of PVRIG on CMV-expanded CD8 T cells: CMV reactive donorswere purchased from Cellular Technology Limited (CTL). Supplied PBMCwere pulsed for 2 hours with 10 uM CMV peptide 494-503 (NLVPMVATV,Anaspec). The PBMC were subsequently washed three times after which theywere plated in 24 well plates for 9 days in RPMI+10% human AB serum(Sigma), glutamax, penicillin/streptomycin, and a cytokine growthcocktail consisting of 2 ng/ml IL-2 (R&D systems) and 10 ng/ml IL-7 (R&Dsystems). After 9 days, non-adherent cells were harvested, phenotypedfor CD8 T cell enrichment, and banked in liquid nitrogen.

To assess expression on CMV-expanded CD8 T cells, binding ofunconjugated CHA.7.518.1.H4(S241P) and CHA.7.538.1.2.H4(S241P) (hIgG4)and their respective controls was assessed in an 8-point titrationseries starting at 666 nM on ice for 30 mins-1 hr. The titration serieswas conducted as a 4-fold serial dilution series. Unconjugated primaryantibodies were detected with an anti-human Fc Alexa 647 conjugatedantibody. Data was analysed using FlowJo and Prism software andcollected on a BD LSR Fortessa X-20.

FACS analysis of cynomolgus PVRIG engineered over-expressing cells: Thefollowing cell lines were used to assess the cross-reactivity ofCHA.7.518.1.H4(S241P) and CHA.7.538.1.2.H4(S241P) with cynomolgus PVRIG(cPVRIG): expi parental and expi cPVRIG over-expressing cells. Thesecells were cultured in DMEM+10% fetal calf serum+glutamax. expi cPVRIGtransient over-expressing cells were generated by electroporating cPVRIGDNA into parental expi cells using the Neon transfection system. ForFACS analysis, expi cPVRIG cells were used between 1-3 days-posttransfection. Parental expi cells were harvested from log growth phase.50,000-100,000 cells of per well of each type were seeded in 96 wellplates. Binding of unconjugated CHA.7.518.1.H4(S241P) andCHA.7.538.1.2.H4(S241P) (hIgG4) and their respective controls wereassessed in an 8-point titration series starting at 10 ug/ml on ice for30 mins-1 hr. The titration series were conducted as a 3-fold dilutionseries. Unconjugated primary antibodies were detected with an anti-humanFc Alexa 647 conjugated antibody. Data was acquired using a FACS CantoII or IntelliCyte and analyzed using FlowJo and Prism software.

Cellular-based receptor-ligand blocking assays: The ability ofCHA.7.518.1.H4(S241P) and CHA.7.538.1.2.H4(S241P) to inhibit theinteraction of PVRIG with its ligand PVRL2 was assessed in a cellularcompetition assay conducted in two orientations.

In the first orientation, PVRL2 is endogenously expressed onun-manipulated HEK cells, and the ability of CHA.7.518.1.H4(S241P) andCHA.7.538.1.2.H4(S241P) to block soluble biotinylated PVRIG Fc bindingto HEK cells was measured. More specifically, biotinylated PVRIG Fcprotein (33 nM) and CHA.7.518.1.H4(S241P) and CHA.7.538.1.2.H4(S241P)(1.03-198 nM, hIgG4) were concominantly added to 100,000 HEK cells andincubated for 1 hour on ice. The extent of biotinylated PVRIG Fc bindingwas subsequently detected by the addition of streptavidin Alexa 647(Jackson Laboratories) for 20-30 minutes on ice. Cells were washed twicein PBS for acquisition using a FACS Canto II. Data was analyzed usingFlowJo, Excel (Microsoft), and Prism.

In the second orientation, HEK cells were engineered to express humanPVRIG (HEK hPVRIG) and the ability of CHA.7.518.1.H4(S241P) andCHA.7.538.1.2.H4(S241P) (hIgG4) to inhibit soluble human PVRL2 Fc wasassessed. More specifically, HEK hPVRIG cells were pre-incubated withCHA.7.518.1.H4(S241P) and CHA.7.538.1.2.H4(S241P) (0.66-66 nM) for 30mins on ice, after which PVRL2 mFc (human PVRL2 with a mouse Fc) wasadded (for 1 hr on ice) and its ability to bind HEK hPVRIG was measured.The extent of PVRL2 mFc binding was detected by the subsequent additionof goat anti-mouse Fc A647 (Jackson Laboratories) for 20-30 mins on ice.Cells were washed twice in PBS for acquisition using a FACS Canto II.Data was analyzed using FlowJo, Excel and Prism.

Cellular-based epitope space analysis: Epitope space forCHA.7.518.1.H4(S241P) and CHA.7.538.1.2.H4(S241P) was assessed on theirability to compete with another for binding to Jurkat cells. Briefly,Jurkat cells were harvested in log growth phase and stained with □□μg/ml unlabeled CHA.7.518.1.H4(S241P) and CHA.7.538.1.2.H4(S241P) for 30mins on ice. Jurkat cells were subsequently spun down, washed, andcounterstained with □ μg/ml Alexa 647-labelled CHA.7.518.1.H4(S241P) andCHA.7.538.1.2.H4(S241P) for 30 mins on ice. The competition of labelledantibodies for PVRIG binding with unlabeled antibodies on Jurkat cellswas assessed by the magnitude of Alexa 647 signal by flow cytometry.Data was acquired using a FACS Canto II and analysed using FlowJo,Excel, and Prism.

Results

CHA.7.518.1.H4(S241P) and CHA.7.538.1.2.H4(S241P) recognize PVRIG onoverexpressing cells, Jurkat cells, and human T cells: The ability ofCHA.7.518.1.H4(S241P) and CHA.7.538.1.2.H4(S241P) humanizedhybridoma-derived PVRIG antibodies to bind to human PVRIG was assessedusing HEK cells that overexpress human PVRIG, Jurkat cells, and primaryT cells. FIG. 20 illustrates the specificity of bothCHA.7.518.1.H4(S241P) (A) and CHA.7.538.1.2.H4(S241P) (B). Bothantibodies bind highly specifically to HEK hPVRIG cells, and do not bindto HEK parental cells.

Binding affinities: Both CHA.7.518.1.H4(S241P) andCHA.7.538.1.2.H4(S241P) also display binding to HEK hPVRIG cells withhigh affinity with their associated Kd values: 0.29 nM forCHA.7.518.1.H4(S241P) and 0.86 nM for CHA7.538.1.2 for binding to HEKhPVRIG cells.

FIG. 21 illustrates the ability of CHA.7.518.1.H4(S241P)(A) andCHA.7.538.1.2.H4(S241P) (B) to bind Jurkat cells that endogenouslyexpress PVRIG. Both are able to bind Jurkat cells with a comparableaffinity to HEK hPVRIG cells.

The affinity of these antibodies to Jurkat cells are 0.15 nM forCHA.7.518.1.H4(S241P) and 0.59 nM for CHA.7.538.1.2.H4(S241P).

FIG. 22 illustrates the ability of CHA.7.518.1.H4(S241P) andCHA.7.538.1.2.H4(S241P) to bind CD8 T cells that were expanded byexposure to CMV peptide (494-503, NLVPMVATV) and endogenously expressPVRIG.

CHA.7.518.1.H4(S241P) and CHA.7.538.1.2.H4(S241P) detect cynomolgusPVRIG (cPVRIG) expressed on expi cells: The ability ofCHA.7.518.1.H4(S241P) and CHA.7.538.1.2.H4(S241P) to bind to cPVRIG wasassessed using expi cells that overexpress cPVRIG. FIG. 23 illustratesthe specificity of both CHA.7.518.1.H4(S241P) (A) andCHA.7.538.1.2.H4(S241P) (B). Both antibodies bind highly specifically toexpi cPVRIG cells, and do not bind to expi parental cells. BothCHA.7.518.1.H4(S241P) and CHA.7.538.1.2.H4(S241P) also display bindingto expi cPVRIG cells with high affinity with their associated Kd valuesof 0.24 nM for CHA.7.518.1.H4(S241P) and 0.58 nM for CHA7.538.1.2.

Cellular-based receptor-ligand blocking assays: The ability ofCHA.7.518.1.H4(S241P) and CHA.7.538.1.2.H4(S241P) to inhibit theinteraction of PVRIG with PVRL2 was assessed in two orientations, asoutlined in the protocols section. In the first permutation, bothCHA.7.518.1.H4(S241P) and CHA.7.538.1.2.H4(S241P) were able tocompletely inhibit the binding of PVRIG Fc to HEK cells (FIG. 24A). TheIC50 values associated with this blocking capacity are 15 nM forCHA.7.518.1.H4(S241P) and 16.1 nM for CHA.7.538.1.2.H4(S241P).Importantly, not all the antibodies derived from the hybridoma campaignconfirmed to bind to PVRIG were able to block the binding of PVRIG Fc toHEK cells. As shown in FIG. 24B, an antibody clone designated CHA.7.544is unable to block the binding of PVRIG Fc to HEK cells.

In the second permutation, both CHA.7.518.1.H4(S241P) andCHA.7.538.1.2.H4(S241P) were also able to completely inhibit the bindingof PVRL2 Fc to HEK hPVRIG cells (FIG. 25A). The IC50 values associatedwith this inhibition are 1.8 nM for CHA.7.518.1.H4(S241P) and 2.53 nMfor CHA.7.538.1.2.H4(S241P). Although the ability ofCHA.7.518.1.H4(S241P) and CHA.7.538.1.2.H4(S241P) were able tocompletely inhibit PVRL2 Fc binding in this permutation, consistent withtheir ability to inhibit PVRIG Fc binding in the first permutation,other antibodies did not show this same trend. More specifically,another humanized hybridoma-derived antibody, CHA.7.530.3, that was ableto completely inhibit binding of PVRIG Fc to HEK cells (firstpermutation, data not shown), was not able to completely inhibit bindingof PVRL2 Fc binding to HEK hPVRIG cells (FIG. 25A). Collectively, thisdata indicates that the second permutation of the cellular-basedreceptor ligand blocking assay is able to distinguish potency ofreceptor-ligand blocking antibodies with more sensitivity compared tothe first permutation. Importantly, CHA.7.544 was shown to be unable toblock the binding of PVRL2 Fc to HEK hPVRIG cells (FIG. 25B) consistentwith its inability to block PVRIG Fc binding to HEK cells.

Cellular-based epitope space analysis: As outlined in the protocolssection, an analysis of the epitope space of CHA.7.518.1.H4(S241P) andCHA.7.538.1.2.H4(S241P) was conducted by assessing their ability tocompete for PVRIG binding. FIG. 26 shows the ability of unconjugatedversions of the antibodies to inhibit binding of the Alexa 647 (A647)conjugated versions of the same antibodies. The data in the FIG. 26depicts the percentage binding of A647 conjugated antibodies relative tothe maximum signal they yield with no competition. The signal yieldedfrom CHA.7.518.1.H4(S241P) A647 and CHA.7.538.1.2.H4(S241P) A647 was notaffected by pre-incubation of the Jurkat cells with isotype control(data not shown). As expected, the signal yielded fromCHA.7.518.1.H4(S241P) A647 and CHA.7.538.1.2.H4(S241P) A647 wassignificantly reduced when in competition with unconjugated versions ofthemselves (data not shown). Interestingly, upon analysis of A647 signalfrom CHA.7.518.1.H4(S241P) and CHA.7.538.1.2.H4(S241P) in the context ofpre-incubation with the unconjugated version of the opposite antibody,there was also significant reduction. This indicates thatCHA.7.518.1.H4(S241P) and CHA.7.538.1.2.H4(S241P) may share a similarepitope space on endogenously expressed PVRIG.

Summary and Conclusions: Mouse versions of anti-PVRIG antibodiesdesignated CHA.7.518 and CHA.7.538 were successfully humanized into ahuman IgG4 isotype (CHA.7.518.1.H4(S241P) and CHA.7.538.1.2.H4(S241P))which retained binding properties towards the human PVRIG antigen. Usingengineered over-expressing cells, Jurkat, and CMV expanded primary CD8 Tcells, CHA.7.518.1.H4(S241P) and CHA.7.538.1.2.H4(S241P) were shown tobe highly specific to endogenous human PVRIG and bound with highaffinity. Furthermore, CHA.7.518.1.H4(S241P) and CHA.7.538.1.2.H4(S241P)also showed reactivity to cyno PVRIG antigen and bound toover-expressing cells with high affinity. Functionally,CHA.7.518.1.H4(S241P) and CHA.7.538.1.2.H4(S241P) were able to inhibitthe interaction of PVRIG with PVRL2 in FACS-based assays. Lastly,CHA.7.518.1.H4(S241P) and CHA.7.538.1.2.H4(S241P) were shown topotentially share epitope space on endogenous human PVRIG due to theirability to compete with one another for binding to Jurkat cells.

F. Example 6: Humanized Antibodies: Functional Analysis of HumanizedAntibodies

The functional activity of several humanized antibodies of the inventionwas validated.

CHO-S OKT3 assay: The CHO-S OKT3 assay was utilized to determine whetherthe humanized PVRIG antibodies, CHA.7.518.1.H4(S241P) andCHA.7.538.1.2.H4(S241P), could enhance CD4+ and CD8+ T cellproliferation, and cytokine secretion. The target cells used in theco-culture assay were the Chinese hamster ovary cell line, CHO-S(ATCC),either stably overexpressing the single chain variable fragment of theanti-human CD3 antibody Clone OKT3 (abbreviated as OKT3), or stablyoverexpressing both OKT3 and human PVRL2 (abbreviated as hPVRL2). CHO-SOKT3 parental cells were grown in serum-free CD-CHO medium (Gibco)supplemented with 40 mM glutamax (Gibco), penicillin/streptomycin(Gibco), and 6 μg/ml puromycin (Gibco). CHO-S OKT3 hPVRL2 cells weregrown in the same CD-CHO medium as the parental cells, but alsosupplemented with 600 μg/ml hygromycin B (Gibco).

Primary CD4+ and CD8+ T cells were isolated from healthy human donorsusing the RosetteSep™ human CD4+ T cell enrichment cocktail (StemcellTechnologies), and the human CD8+ microbeads (Miltenyi Biotec),respectively, and frozen in liquid nitrogen. On the day of theco-culture assay, CD4+ or CD8+ T cells were thawed, counted, and labeledwith 1 μM CFSE (Life Technologies) for 10 minutes at 37° C. Followingthis incubation, T cells were washed and resuspended in complete mediumcontaining RPMI (Gibco), supplemented with 10% heat-inactivated FBS,glutamax, penicillin/streptomycin, non-essential amino acids (Gibco),sodium pyruvate (Gibco), and 50 μM β-mercaptoethanol (Gibco). CHO-S OKT3and CHO-S OKT3 hPVRL2 cells were harvested from culture, and treatedwith mitomycin C (Sigma-Aldrich) for 1 hour at 37° C. with periodicmixing. After the incubation, the target cells were thoroughly washed,counted, and resuspended in complete RPMI medium. The assay was set upwith a 5:1 ratio of T cells (100,000) to target cells (20,000). Thetarget cells, T cells, and 10 μg/ml of each antibody treatment wereadded together in a 96-well U-bottom plate (Costar), and incubated foreither 3 days (CD8+ T cells), or 5 days (CD4+ T cells) at 37° C. ThePVRIG antibody treatments included human CHA.7.518.1.H4(S241P) IgG4,human CHA.7.538.1.2.H4(S241P) IgG4, human CHA.7.530.3 IgG4 (partialreceptor/ligand blocking antibody), and mouse CHA.7.544 IgG1(non-receptor/ligand blocking antibody). In addition to the PVRIGantibodies, the activity the mouse anti-human DNAM-1 IgG1 (Clone DX11,BioLegend), mouse IgG1 isotype control (Clone MOPC21, BioLegend), and ahuman IgG4 isotype control was also assessed. For antibodydose-titrations, 3-fold dilutions from 66 nM to 0.264 nM of the PVRIGantibodies, and the respective isotype control antibody were utilized.

After the 3 or 5-day incubation period, co-culture supernatants wereanalyzed for secreted cytokines, including IL-2, IL-4, IL-5, IL-6, IL-9,IL-10, IL-13, IL-17A, IL-17F, IL-21, IL-22, TNFα, and IFNγ, with thecytometric bead array (CBA) human Th1/Th2/Th17 cytokine kit (BDBiosciences), or with the LEGENDplex™ Human Th cytokine kit (BioLegend).T cell proliferation was measured by staining CD4+ or CD8+ T cells withthe LIVE/DEAD fixable aqua dead cell stain kit (ThermoFisherScientific), anti-CD4 antibody (Clone RPA-T4, BioLegend), and anti-CD8antibody (Clone HIT8a, BioLegend), and gating on the percentage of live,CFSE low proliferating CD4+ or CD8+ T cells. Data was acquired using aFACS Canto II (BD Biosciences), and analyzed using FlowJo (Treestar) andPrism (Graphpad) software.

Results

CHA.7.518.1.H4(S241P) and CHA.7.538.1.2.H4(S241P) Enhance CD4⁺ T CellProliferation in a hPVRL2-Dependent Manner:

The ability of the CHA.7.518.1.H4(S241P) and CHA.7.538.1.2.H4(S241P)humanized hybridoma-derived PVRIG antibodies to enhance primary CD4+ Tcell proliferation in vitro was assessed with the CHO-S OKT3 assay. FIG.27A shows the percentage proliferating CD4+ T cells from arepresentative donor in response to co-culture with the CHO-S OKT3hPVRL2 target cells and different PVRIG antibodies. In this donor,humanized CHA.7.518.1.H4(S241P) and CHA.7.538.1.2.H4(S241P) antibodiesincrease CD4+ T cell proliferation compared to the human IgG4 isotypecontrol (dashed line). The partial receptor/ligand blocking antibody,human CHA.7.530.3 IgG4 only weakly enhances T cell proliferation, whilethe non-receptor/ligand blocking antibody, mouse CHA.7.544 IgG1 has noeffect compared to the isotype control antibodies. The anti-DNAM-1antibody reduces CD4+ T cell proliferation. FIG. 27B demonstrates thatthe effects of the humanized CHA.7.518.1.H4(S241P) andCHA.7.538.1.2.H4(S241P) PVRIG antibodies, and the anti-DNAM-1 antibodyare dependent on hPVRL2 overexpression on the target cells. FollowingCHA.7.518.1.H4(S241P) and CHA.7.538.1.1 antibody treatment, a greaterincrease in CD4+ T cell proliferation is observed when the CD4+ T cellsare co-cultured with the CHO-S OKT3 hPVRL2 cells, compared to co-culturewith the CHO-S OKT3 parental cells. Similarly, the anti-DNAM-1 antibodyonly decreases CD4+ T cell proliferation when T cells are co-culturedwith the hPVRL2-expressing CHO-S OKT3 cells.

CHA.7.518.1.H4(S241P) and CHA.7.538.1.2.H4(S241P) enhance CD8+ T cellproliferation and IFN-g secretion: FIG. 28A-B illustrate the ability ofhumanized PVRIG antibodies, CHA.7.518.1.H4(S241P) andCHA.7.538.1.2.H4(S241P), to increase CD8+ T cell proliferation in theCHO-S OKT3 assay. In two different human CD8+ T cell donors,CHA.7.518.1.H4(S241P) and CHA.7.538.1.2.H4(S241P) antibodies increaseCD8+ T cell proliferation compared to the human IgG4 isotype controlwhen T cells are co-cultured with the CHO-S OKT3 hPVRL2 cells. However,the mouse CHA.7.544 IgG1 has little to no effect. As observed with theCD4+ T cells, the anti-DNAM-1 antibody reduces CD8+ T cellproliferation. FIG. 28C shows that the humanized PVRIG antibodies,CHA.7.518.1.H4(S241P) and CHA.7.538.1.2.H4(S241P) also enhance IFNγsecretion in the CHO-S OKT3 assay. In three different human CD8+ T celldonors, CHA.7.518.1.H4(S241P) and CHA.7.538.1.2.H4(S241P) antibodiesincrease IFNγ production compared to the human IgG4 isotype control(dashed line). Increases in IL-10, IL22 and TNFα were also observedfollowing CHA.7.518.1.H4(S241P) and CHA.7.538.1.2.H4(S241P) antibodytreatment (data not shown).

CHA.7.518.1.H4(S241P) and CHA.7.538.1.2.H4(S241P) consistently enhanceCD4+ T cell proliferation across multiple human donors: Next, todemonstrate that the CHA.7.518.1.H4(S241P) and CHA.7.538.1.2.H4(S241P)antibodies could reproducibly enhance T cell function, the effects ofthe humanized PVRIG antibodies on CD4+ T cell proliferation wereexamined across 11 different donors in the CHO-S OKT3 assay. FIG. 29demonstrates that both CHA.7.518.1.H4(S241P) and CHA.7.538.1.2.H4(S241P)consistently increased CD4+ T cell proliferation in the majority of thetested donors compared to the human IgG4 isotype control antibody when Tcells were co-cultured with the CHO-S OKT3 hPVRL2 cells. Furthermore,the partial receptor/ligand blocking antibody, CHA.7.530.3, and thenon-receptor/ligand blocking antibody, CHA.7.544, do not consistentlyenhance CD4+ T cell proliferation across the same donors.

CHA.7.518.1.H4(S241P) and CHA.7.538.1.2.H4(S241P) have a dose-dependenteffect on CD4+ and CD8+ T cell proliferation: Finally, thedose-dependent effect of the humanized PVRIG antibodies,CHA.7.518.1.H4(S241P) and CHA.7.538.1.2.H4(S241P) was measured in theCHO-S OKT3 assay. Decreasing the dose of the CHA.7.518.1.H4(S241P) andCHA.7.538.1.2.H4(S241P) antibodies lowers the percent of CD4+ T cell(FIG. 30A), and CD8+ T cell (FIG. 30B) proliferation when the T cellsare co-cultured with the CHO-S OKT3 hPVRL2 cells. This dose-dependenteffect on T cell proliferation is not observed with the CHA.7.544antibody, nor the IgG4 isotype control. Furthermore, no biphasic effectwith the dose titration was observed, suggesting a lack of agonistactivity of the humanized PVRIG antibodies.

Summary and Conclusions

Humanized PVRIG antibodies, CHA.7.518.1.H4(S241P) andCHA.7.538.1.2.H4(S241P), had in vitro functional activity in the primarycell-based CHO-S OKT3 assay. CHA.7.518.1.H4(S241P) andCHA.7.538.1.2.H4(S241P) both increased CD4+ and CD8+ T cellproliferation in a dose-dependent manner. CHA.7.518.1.H4(S241P) andCHA.7.538.1.2.H4(S241P) were also capable of augmenting IFNγ secretionin the CHO-S OKT3 assay. It was shown that the activity of theCHA.7.518.1.H4(S241P) and CHA.7.538.1.2.H4(S241P) antibodies wasdependent on overexpression of hPVRL2 on target cells.CHA.7.518.1.H4(S241P) and CHA.7.538.1.2.H4(S241P) consistently enhancedT cell activity across multiple human donors, while the non-blockingCHA.7.544 antibody had little to no effect.

G. Example 7: Development of Rat Monoclonal Antibodies for Mouse PVRIG

Development of rat monoclonal antibodies (mAbs) was performed atAldevron Freiburg (Germany). Antibodies against mouse PVRIG protein wereraised by using DNA immunization technology. Immunization vectorexpressing mouse PVRIG introduced into the host organism (rat). MousePVRIG was expressed, and an immunization response was generated.Positive antisera identification and hybridomas screening were analyzedon cells transiently express mouse PVRIG.

Rat Anti-Mouse PVRIG pAb Generation

Development of rat polyclonal antibodies against mouse PVRIG proteinincluded cloning of mouse PVRIG extracellular domain into Aldevronproprietary immunization vector, and cloning of the full length and theextracellular domain into Aldevron proprietary screening vectors. Thevarious expression vectors used for immunizations and for screening wereconfirmed by FACS on cells transiently express mouse PVRIG. Three ratswere then immunized with the immunization vector. Immune sera were takenand diluted sera were tested by FACS using cells transiently transfectedwith the screening vectors. Production bleeds from each rat werecollected, and purification using protein-A was performed.

Rat Anti-Mouse PVRIG mAb Generation

Fusion of rat lymphocytes and selection using Aldevron's test systemswere performed. This included: Fusion of 20×96-well plates followed byinitial screening by Cell Based ELISA (cELISA), on transientlytransfected cells with mouse PVRIG ECD (extracellular domain) or FL(full length). 108 positive clones (bound to cells expressing mousePVRIG ECD\ FL) were further propagated and retested. 30 positives cloneswere propagated into T-25 flasks and the supernatants were tested incell based ELISA. 23 hybridoma clones were selected for furthersubcloning. Serum free supernatant was tested by cELISA and by FACS.Total of 21 clones were generated and binding was confirmed on cellsover expressing the mouse PVRIG protein.

Abs Characterization

Binding of the rat anti mouse PVRIG test bleeds, purified pAb,pre-clonal and clonal supernatants as well as the purified mAbs, wasanalyzed by Flow Cytometry, using stable HEK293 cells over expressingthe mouse PVRIG. The binding of the antibodies to D10.G4.1 cellsendogenously expressing mouse PVRIG was also tested. Specific cellsurface expression of mouse PVRIG was confirmed. Cells (1-2×10⁵) werestained with Fixable viability stain diluted 1:1000 in PBS, for 10 minat R.T. followed by cells washing with PBS. The Abs were then added tocells (diluted in FACS buffer) followed by staining with goat antirat-PE (diluted 1:100 in FACS buffer).

mAbs specificity was tested by siRNA for PVRIG transfection of D10.G4.1cell line endogenously expressing mouse PVRIG. Reduction in cell surfacewas observed following mouse PVRIG knockdown.

mAbs Binding to NK Cells was Also Tested by FACS.

Binning Assay was Performed to Demonstrate mAbs Diversity.

Affinity of the purified mAbs (Kd) was determined by FACS titration onstable cells over expressing the mouse PVRIG versus empty vectortransduced cells, and on D10.G4.1 cell line. Cells (1×10⁵) were stainedwith Fixable viability stain diluted 1:1000 in PBS, for 10 min at RTfollowed by cells washing with PBS. The Abs were then added to cells (8concentrations-series dilution 1:3, 10-0.01 μg/ml diluted in FACSbuffer) followed by staining with Goat Anti rat-PE (diluted 1:100 inFACS buffer).

mAbs Characterization—Summary Table

Table 7 (columns 1-10) summarize the data generated for thecharacterization of the anti-mouse PVRIG antibodies.

-   -   Column 1 represent the Ab code ID    -   Column 2 represent the Ab name provided by Aldevron    -   Column 3 represent FACS data as MFI ratio on stable over        expressing cells over empty vector transduced cells at 10 μl/ml        mAb concentration    -   Column 4 represent affinity (nM) on the over expressing HEK        cells    -   Column 5 represent binding to NK cells at 10 ug\ml mAb        concentration    -   Column 6 represent MFI ratio of binding of D10.G4.1 cell line        over isotype control    -   Column 7 represent affinity (nM) to D10.G4.1 cell line    -   Column 8 represent the various bins in the epitope binning assay    -   Column 9 represent % Receptor-Ligand blocking assay (mouse        PVRIG-Fc fusion protein binding to mouse PVRL2 over expressing        cells) and IC50 (nM)    -   Column 10 represent % Receptor-Ligand blocking assay (mouse        PVRL2-Fc fusion protein binding to mouse PVRIG over expressing        cells) and IC50 (nM)

AB-406 and AB-407 demonstrated blocking activity in both Receptor-Ligandbinding assays have relative high affinity, binds to NK and to D10.G4.1cells.

These Abs were selected for TME expression and for in vivo studies.

TABLE 7 Anti-mouse PVRIG monoclonal Abs characterization. MFirExpression Kd (nM) % R-L (HEK Kd (nM) Expression in D10.G4.1 On % R-Lblocking OX/EV) On OX in NKMFir MFir D12.G4.1 Epitop (029-Fc)(mPVRL2-Fc) LIMS ID Ab name 10 ug/ml Cells (Ab/Iso) (Ab/Iso) CellsBinning (IC50) (IC50) AB-400 BOJ-1F11-H6 10 0.1393 NT 13.2 35.68  1Agonist Agonist AB-401 BOJ-3E2-F4 55.7 2.4 NT 7.5 NT 1 Agonistinconsistent AB-402 BOJ-4F11-H6 4.8 0.08974 NT 20 NT 1 Agonist AgonistAB-403 BOJ-4G1-E3 28 57.64 — 2 NT 4 78%(2.782) Inconsistent AB-404BOJ-4H8-E3 18.6 0.386833 1.5 6.8 NT 1 Inert 93-98%  AB-405 BOJ-5A4-E313.5 0.08871 NT 6.7 4.596 1 Agonist Agonist AB-406 BOJ-5C7-B3 54.51.884667 2.2 10 8.577 3 77%(3.679) 97-100% AB-407 BOJ-5G4-F4 50 0.3344272.8 8 1.325 3 95%(3.992) 95-100% AB-408 BOJ-8G1-G1 18.9 5.098 1.7 8 NT 490%(3.585) Agonist AB-409 BOJ-9B1-D9 24.8 0.1555 NT 10 NT 1 Agonistinconsistent AB-410 BOJ-11C2-G9 16.6 0.2218 2.4 6.8 NT 1 Agonistinconsistent AB-411 BOJ-12E2-F8 36 5.3405 NT 5.4 NT 3 94%(4.311)95-100%(1.189) AB-412* BOJ-14H2-F4 3.3* NT NT 2 NT 2 Agonist 62-67%(23.87) AB-413* BOJ-15B3-E11 49.3 1.09345 2.1 12 4.693 373%(2.836) 95-100%(0.58)  AB-414 BOJ-15F8-C6 24.8 2.0395 1.6 8.5 16.48 3 90%(2.907) 95-100%(1.164) AB-415 BOJ-16E7-G8 24 17.573 2.2 5 NT 595%(6.727) 93-100%(5.643) AB-416 BOJ-17C4-D4 26.3 6.357 1.5 5 NT 1 Inert 92-94%(1.963) AB-417* BOJ-17C7-H5 15 59.16 — 2.5 NT 4 72%(3.455) InertAB-418 BOJ-18C1-C10 64.8 62.98 NT 4.4 NT 5 94%(18.18)    100%(8.055)AB-419 BOJ-18D2-F5 17 0.3169 1.5 6.3 NT 1 Agonist 57-64%  AB-420BOJ-19D9-C7 33.6 3.172 1.2 20 NT 4 78%(9.805) Agonist

H. Example 8: Combination Testing with Additional Immune CheckpointInhibitors

Background

While antibody blockade of the CTLA4 and PD1 pathways has emerged as aneffective treatment modality for cancer, the majority of patients do notderive long term benefit, suggesting a need for targeting of additionalimmune checkpoints. Employing our unique computational algorithms todefine new members of the B7/CD28 family we identified PVRIG, which isexpressed by multiple subsets of T and NK cells. We report here itsexpression pattern, functional characterization, and anti-tumor activityof blocking antibodies targeting this molecule.

Methods

Utilizing the Predictive Discovery platform PVRIG was identified as apotential novel immune checkpoint, after which a retroviral cellscreening library was used to identify its cognate binding counterpart.Target effects on T-cell modulation were assessed with primary andtumor-derived T-cell assays, taking advantage of target overexpression,knockdown, and antagonist antibody approaches. Antibodies against thehuman protein were screened for their ability to enhance T-cellactivation in vitro, while antibodies targeting the mouse orthologuewere assessed in vivo for effects on tumor growth inhibition insyngeneic models

Results

A PVRIG-Fc-fusion protein was found to bind PVRL2, with bindingspecificity confirmed both by ELISA and flow cytometry analysis. PVRIGdemonstrated unique expression kinetics upon T-cell activation, withdetection of the target on memory T-cells, as well as on NK cells and γδT-cells. A panel of high affinity human antibodies with the ability toblock interaction of PVRIG with PVRL2 were generated, which when testedin vitro were shown to enhance activation of both primary CD4+ andtumor-derived CD8+ T-cells through a PVRL2-dependent mechanism.

Since CHA.7.518.1.H4(S241P) is not mouse cross-reactive, in vivo studieswere conducted with a surrogate blocking anti-mouse PVRIG antibody. Whencombined with anti-PDL-1 blockade, anti-mouse PVRIG inhibits growth ofestablished tumors in both the CT26 and MC38 colorectal cancer models.Combination testing with additional immune checkpoint inhibitors, aswell as in PVRIG knockout mice, is ongoing

Conclusions

High affinity antagonistic antibody, is able to enhance human T-cellactivation, and a surrogate antibody with similar characteristics showssynergy with PD-L1 in vivo in multiple syngeneic models. Overall, ourdata demonstrates the utility of targeting PVRIG in addition to other B7family checkpoints for the treatment of cancer

I. Example 9: In Vivo POC Study: Efficacy of Anti MPVRIG Mabs in CT26Tumor Model

This example describes the efficacy of anti mPVRIg mAbs treatment inCT26 murine colon carcinoma model as mono-therapy or in combination withanti-PDL-1 treatment.

Materials and Methods

Tumor Challenge Experiments:

CT26 colon carcinoma was purchased from ATCC (CRL-2638). Cells werecultured in RPMI 1640 (Biological Industries, 01-100-1A) with 10% FBS(Biological Industries, 04-127-1A), and 100 μg/mLpenicillin/streptomycin (Biological Industries, 03-031-1B). For tumorimplantation, cells were harvested and washed, counted and suspended to10⁷ cells/ml in cold RPMI 1640 and placed in ice. BALB/c mice ((female,8 wk) Envigo), were anesthetized with 10% Ketamine (Clorketam; SAGARPAQ-7090-053) and 10% Xylazine (Sedaxylan; BE-V254834) mixture injectedintraperitoneal. Next, the back of the mice was shaved and disinfectedwith a 70% Ethanol solution. Tumor cells were injected as 50 μl of 5×10⁵CT26 cells subcutaneously into the back right flank of mice. The mAbadministration started at day 4 (Mono treatment) or day 7 (Combotreatment) post tumor inoculation when tumors were at volume of 30-50mm³ (Mono treatment) or reached the volume of 60-90 mm³ (Combotreatment); and was given intra-peritoneal (i.p.) in a finalvolume/injection of 200 ul, for 3 wks for a total of 6 administrations.Tumor growth was measured with electronic caliper every 2-3 days and wasreported as 0.5×W²×L mm³. Mice were sacrificed with CO2 at either studytermination or any of the following clinical endpoints: tumor volume≥2250 mm³, tumor ulceration, body weight loss ≥20%, or moribundappearance

Antibodies:

The chimeric anti-mouse PVRIg antibodies (mAb 406 and mAb 407) used inthis study, engineered as a Rat IgG2b isotype monoclonal antibody (mAb)were shown to bind to 293HEK transfectants expressing mPVRIg and blockbinding of mPVRL2 to these cells. The mIgG1 anti-mouse PDL-1 inhibitorused in this study was mAb YW243.55.S70. The YW243.55.S70 antibody is ananti-PD-L1 described in WO 2010/077634 (heavy and light chain variableregion sequences shown in SEQ ID Nos.20 and 21, respectively, of WO2010/077634), and having a sequence disclosed therein.

All mAbs were formulated in sterile PBS and were low in endotoxin (<0.05EU/mg).

TABLE 8 Tested mAbs. 1 Mouse IgG1, k Isotype Ctrl. BP0083 BioXcell(MOPC-21) 2 Rat IgG2b, k Isotype Ctrl. BP0090 BioXcell (LTF-2,) 3Benchmark anti PDL-1 YW243.55.S70 Compugen inc. (mIgG1) 4 Anti CGENPVRIG mAb 406 BOJ-5C7-B3 ALDEVERON (Rat IgG2b) 5 Anti CGEN PVRIG mAb 407BOJ-5G4-F4 ALDEVERON (Rat IgG2b)Study DesignMono Treatment

Eight weeks old BALB/c female mice were purchased from Envigo andmaintained in an SPF animal facility for 1 week prior to beginning theexperiment. Mice were anesthetized, shaved and inoculated subcutaneouslywith 50 μl of 5×10⁵ CT26 tumor cells. At day 4 post tumor inoculation,mice were randomly assigned into treatment groups of n=10 (as describedbelow). Mice were treated with mAbs (as detailed below) injected on day4, 7, 11, 14, 18 and 21 post inoculation. Tumor growth was measured withcaliper every 2-3 days.

TABLE 9 Treatment groups. # Dose # Vol/Dose Group Treatment/mAb (mg/Kg)Dose (ul) 1 Vehicle 6 200 2 mIgG1 iso Ctrl 5 6 200 3 Rat IgG2b iso Ctrl10 6 200 4 Anti-PDL-1 mIgG1 5 6 200 5 Anti-mPVRIg mAb 406 rIgG2b 10 6200 6 Anti-mPVRIg mAb 407 rIgG2b 10 6 200Combo Treatment

For Combo of anti-mPVRIg and anti-mPDL-1 mAbs treatments. Mice weretreated as described in the Mono treatment. At day 7 post tumorinoculation, mice were randomly assigned into treatment groups of n=10as described below. Mice were treated with mAbs (as detailed below)injected on day 7, 11, 14, 18, 21 and 25 post tumor inoculation.

TABLE 10 Treatment dosages. Vol/ # Treatment/ Dose Treatment/ Dose #Dose Group mAb 1 (mg/Kg) mAb 2 (mg/Kg) Dose (μl) 7 mIgG1 5 Rat IgG2b 106 200 iso Ctrl iso Ctrl 8 Anti- 5 Rat IgG2b 10 6 200 PDL-1 iso CtrlmIgG1 9 Anti- 5 Anti- 10 6 200 PDL-1 mPVRIg mIgG1 mAb 406 rIgG2b 10Anti- 5 Anti- 10 6 200 PDL-1 mPVRIg mIgG1 mAb 407 rIgG2bStatistical Analysis:

Two-way ANOVA with repeated measures, followed by two way ANOVA withrepeated measures for selected pairs of groups using JUMP (StatisticalDiscoveries TM) software. Analyses of tumor growth measurements wereperformed by comparing tumor volumes measured on the last day on whichall study animals were alive. Statistical differences in percentage ofmice tumor free were determined by a Log Rank Mantel-Cox test. Values ofP<0.05 were considered significant.

* p<0.05; ** p<0.01; *** p<0.001. For each experiment, the number ofreplicates performed and the number of animals per group are describedin the corresponding figure legend(s) (FIGS. 47-48).

Results

Monotherapy Activity of Anti-mPVRIg and Anti-mPDL-1 in Syngeneic CT26Tumor Model

We began preclinical assessment of anti-mPVRIg and anti-mPDL-1monotherapy in mouse syngeneic C126 tumor model. We treated mice with amIgG1 isotype anti-PDL-1 antibody (YW243.55.S70) or with rIgG2b isotypeanti-mPVRIg (mAbs 406 and 407).

In a semi-therapeutic treatment model of C126 colon carcinoma,monotherapy with anti-PDL-1 was significantly efficacious (P<0.0001),eliciting a 70% of TGI compared to control mIgG1 isotype, greater ratesof tumor rejection with rapid tumor rejection and durable antitumorimmunity observed in a majority of mice (FIG. 63A+B).

Groups treated with either anti-mPVRIg mAb 406, and anti-mPVRIg mAb 407showed similar tumor growth rates with no TGI over rIgG2b isotype (FIG.63A+B). Accordingly, anti-PDL-1 mIgG1 treatment prolonged the survivalof mice (P<0.01, FIG. 63C), with 5 out of 10 individuals demonstrating acomplete tumor clearance (FIG. 63B). No effect of anti-mPVRIg mAbs onsurvival rates was observed.

Activity of Anti-PVRIg and Anti-PDL-1 Combination in Syngeneic MouseTumor Model

Next, we assessed the activity of anti-PVRIg combination therapy inmouse syngeneic tumor model.

In a therapeutic treatment model of CT26 colon carcinoma, administrationof anti-PDL-1 with control rIgG2b isotype treatment, initiated on day 7post inoculation, was not efficacious, while combination of anti-PVRIgmAb 407 with anti-PDL-1 elicited significant TGI (56%, P=0.0005), higherrates of tumor rejection with 4 out of 10 individuals demonstrating acomplete tumor clearance (FIG. 64A+B) and promoted better antitumoractivity, with durable antitumor immunity detected (P<0.01, FIG. 64C).Combination of anti-PVRIg mAb 406 with anti-PDL-1 was partiallyefficacious, resulting a 33% of TGI, however, the anti-tumor responserecorded was transient and no effect on survival rate was observed.

Conclusions

The mPVRIg was predicted to play a role as a novel B7-like molecule andthus as a potential target for antibody based cancer immunotherapy.Several human in vitro experimental systems have demonstrated animmune-modulatory effect for mPVRIg. In the studies presented in thisreport we have evaluated the in vivo anti-cancer effect of mAbs directedagainst mPVRIg. In our study, treatment with 10 mg/kg (200 ug/mouse) ofanti-mPVRIg as monotherapy in a minimal disease set-up, i.e. treatmentinitiation on day 4 (tumor mean of 40 mm³), did not result in TGI orsurvival advantage while positive control anti-PDL-1 mAb exhibitedsignificant TGI and resulted prolonged survival.

Anti-mPVRIg mAbs were tested also in combination with anti-PDL-1treatment. Treatment with 10 mg/kg (200 ug/mouse) was initiated on day7, when tumors reach an average size of 75 mm³. Combination therapy ofAnti-mPVRIg mAb 407 with anti-PDL-1 in therapeutic CT26 model exhibitedtumor growth inhibition and prolonged survival of treated mice. Theeffect on tumor growth varied between individual mice with someindividuals demonstrating a complete tumor clearance while otherindividuals exhibiting partial response (transient TGI) and someindividuals were not responsive. An in vivo effect of anti-mPVRIg andanti-mPDL-1 combination treatment was also shown in MC38 andB16-Db/gp100 tumor models (data not shown).

Additional in vivo studies are planned to assess dose dependencies andefficacy in additional syngeneic models or in combination withadditional treatment compounds or regimens.

J. Example 10: Tigit Therapeutic Antibody Discovery by Phage Display

1. Introduction

A phage display antibody discovery campaign was conducted to isolatehuman TIGIT binders from a naïve human fab library using recombinanthuman TIGIT extra-cellular domain as target antigen. Forty-five novelhuman TIGIT-specific antibodies were isolated and generated as humanIgG4, inclusive of an optional S241P in the hinge region as discussedherein. The resulting antibodies were screened for their ability toblock the TIGIT-PVR interaction and for cross-reactivity withcell-expressed cynomolgus TIGIT by flow cytometry. Two of theseantibodies were further optimized for higher human and cynomolgus TIGITbinding affinity.

2. Protocols

Antigens for Antibody Discovery by Phage Display:

Two formats of human TIGIT protein were used as antigens in phagedisplay. The first comprised of the human TIGIT ECD (Met22-Pro141) fusedto a C-terminal polyhistidine tag (hTIGIT-HIS) and was either generatedin-house or sourced commercially from Sino Biological Inc. The secondantigen format comprised of the human TIGIT ECD fused to a human IgG1 Fcdomain at the C-terminus (hTIGIT-hFc) and was either generated in-houseor sourced commercially from R&D Systems.

Functional QC of Antigens:

The recombinant TIGIT antigens used for biopanning were functionallyvalidated by their ability to bind to human PVR, the ligand of humanTIGIT. Biotinylated antigens were tested for PVR binding, either byELISA or flow cytometry. Biotinylated hTIGIT-HIS was validated by itsability to bind hPVR-hFc (Sino Biological Inc.) by ELISA. BiotinylatedhTIGIT-hFc was validated by flow cytometry for its ability to bindendogenously surface expressed PVR on Expi293 cells.

Phage Panning of Human Antibody Library:

Two phage campaigns, utilizing either human TIGIT-HIS (campaign 1) orhuman TIGIT-hFc (campaign 2) as antigens, were executed. Panningreactions were carried out in solution, using streptavidin-coatedmagnetic beads to display the biotinylated TIGIT antigens. Bothcampaigns used a human fab antibody phage display library for initialdiscovery. Three rounds of panning were carried out using the respectivehuman TIGIT antigens, with higher wash stringency and lower antigenconcentrations in each successive round of panning. Antibody CPA.9.002,generated in campaign 1, was optimized for improved human TIGIT bindingby generating a phage library by saturation mutagenesis of L-CDR3 andpanning the resulting library against human TIGIT-HIS (campaign 3). Twoantibodies, CPA.9.059 and CPA.9.027, generated in campaigns 2 and 3,respectively, were also optimized for improved human TIGIT affinity andcyno TIGIT cross-reactivity (campaign 4). For each antibody, a phagelibrary was generated by saturation mutagenesis of two CDRs (anycombination of H-CDR1, H-CDR2, H-CDR3, L-CDR1, or L-CDR3). The resultingphage libraries were panned for four rounds against human TIGIT-HIS andC-terminal HIS-tagged cyno TIGIT ECD recombinant protein in alternatingrounds of panning. The panning antigens used were as follows: 1 nM humanTIGIT-HIS in round 1, 1 nM cyno TIGIT-HIS in round 2, 0.1 nM humanTIGIT-HIS in round 3, and 0.1 nM cyno TIGIT-HIS in round 4.

Binding Screens Using Antibodies Expressed as Fab Fragments:

The phagemid construct contains an amber stop codon that allows it tofunction as a fab expression vector. Transformation of these vectorsinto E. coli and induction with isopropyl β-D-1-thiogalactopyranoside(IPTG) results in periplasmic expression of soluble fab molecules. Fabproteins secreted into the E. coli periplasm were extracted by osmoticshock for binding screens.

Primary Screen by ELISA:

The fab PPE extracts were tested for binding to the panning antigenhTIGIT-HIS or hTIGIT-hFc by ELISA. The positive hits from the ELISAscreen were sequenced using heavy chain and light chain-specificprimers. The sequences were assembled and analyzed. Clones were deemedsequence-unique if there were more than one non-conservative differencesin heavy chain CDR3.

Secondary Screen by Flow Cytometry:

The sequence-unique ELISA-positive fab clones were selected and analyzedfor their ability to bind human TIGIT over-expressing Expi293 cells byflow cytometry. Parental Expi293 cells were used as a negative controlfor each fab sample.

Re-Formatting of Fab Hits and Production as Human IgG4 Molecules:

Potential human TIGIT binding fabs were converted to full length humanIgG4 (including a S241P hinge mutant, see Aalberse et al., Immunology202 105:9-19, hereby incorporated by reference in its entirety, and inparticular for the discussion of S241P and references 1, 2 and 3 citedtherein) for further characterization. Protein expression constructswere derived by PCR-amplification of variable heavy and light chainsequences, which were sub-cloned into pUNO3 vector (Invivogen).

3. Results

Functional QC of the Human TIGIT Recombinant Proteins:

The hTIGIT-HIS and hTIGIT-hFc recombinant proteins, either generatedin-house or sourced commercially, were functionally validated by theirability to bind to human PVR. Human PVR (Fc-conjugated) showed adose-dependent binding to biotinylated hTIGIT-HIS in ELISA (data notshown). Similar binding was observed in the reverse orientation wherePVR was immobilized on the ELISA plate and hTIGIT-HIS was in solution(data not shown).

The hTIGIT-hFc protein was functionally validated by binding to PVR in aflow cytometry assay. In this assay, the hTIGIT-hFc protein was titratedagainst Expi293 cells that endogenously express human PVR. Theinteraction was detected using an anti-hFc secondary antibody conjugatedto AF647 fluorescence label. An irrelevant Fc protein was used as acontrol (data not shown).

Functional assays were done on a number of the candidates as isdescribed in the Examples below.

Affinity Maturation Binding Screens Using Antibodies Expressed as FabFragments:

Eight 96-well plates of periplasmic extracted fab clones were analyzedfor the de novo campaigns (1 and 2). Seventy-three unique clones wereidentified in campaign 1 using the hTIGIT-HIS protein as target antigen.Secondary screening of the 73 ELISA positive clones by flow cytometryidentified 21 positive for binding to human TIGIT over-expressingExpi293 cells. A similar screen for campaign 2 (hTIGIT-hFc as targetantigen) yielded 37 ELISA-positive clones, 24 of which were alsopositive for binding to human TIGIT over-expressing Expi293 cells, byflow cytometry (FIG. 52).

Two 96-well plates of fab clones (as PPEs) were screened for theoptimization/affinity maturation campaigns (3 and 4). The ELISA-positiveunique variants were screened for binding to human and/or cynomolgusTIGIT over-expressing Expi293 cells in flow cytometry. The bindingaffinities of the top clones to the hTIGIT-HIS protein was alsoevaluated by Surface Plasmon Resonance (SPR). The first cycle ofaffinity maturation of CPA.9.002 antibody yielded 5 new antibodies,CPA.9.021, CPA.9.027, CPA.9.044, CPA.9.048, and CPA.9.049, withmutations in the L-CDR3 and at least 3-fold improvement in the bindingaffinity for recombinant human TIGIT. A second cycle of optimization ofCPA.9.027 antibody yielded 4 new antibodies with at least 25-foldimprovement in binding to recombinant human TIGIT. The new variantsshowed mutations in the H-CDR2 and L-CDR3 (CPA.9.083 and CPA.9.086) andadditionally in the L-FR4 for CPA.9.089 and CPA.9.093. Optimization ofCPA.9.059 resulted in two new antibodies, CPA.9.101 and CPA.9.103, withsignificantly improved binding to cynomolgus TIGIT as well as asignificant improvement in the human TIGIT binding for CPA.9.103. Themutations were observed in H-CDR3 and L-CDR1 for both the new variants.Additionally, minor changes in L-FR1 were observed for CPA.9.101.

Reformatting of the ELISA and FACS Positive Fabs into hIgG4:

Forty-five unique fabs positive for ELISA and flow cytometry human TIGITbinding were reformatted for expression as human IgG4 molecules,inclusive of an optional S241P hinge variant as discussed herein. Inaddition, 11 affinity optimized variants were also reformatted as IgG4.The sequences of selected phage-derived antibodies are shown in FIG. 53.The sequences of two benchmark antibodies, BM26 (WO2016/028656, Clone3106) and BM29 (US2016/0176963, Clone 22G2) are also shown in FIG. 53for comparison. The reformatted antibodies were evaluated for binding tohuman TIGIT over-expressing Expi293 cells and a binding curve wasgenerated to calculate the equilibrium binding constant (KD). Theseantibodies were also evaluated for binding to cyno TIGIT over-expressingExpi293 cells as well as their ability to block the interaction betweenhuman TIGIT and human PVR in cell-based assays. Based on thesecharacterization, a subset of these antibodies were selected for invitro functional assays as more fully described below.

K. Example 11: Tigit Therapeutic Antibody Discovery by Hybridoma

1. Rationale and Objectives

Hybridoma technology using known and standard methods in the field wasused to generate murine antibodies that bind to human TIGIT with highaffinity, are cross-reactive with non-human primate (cynomolgus macaque,Macaca fascicularis, referred to as cyno) TIGIT, and block theinteraction of TIGIT with its ligand, PVR (CD155).

2. Summary

Balb/c mice were immunized with recombinant forms of human and cynoTIGIT extra-cellular domain proteins. Cells isolated from the spleen andlymph nodes of immunized mice were fused with the Sp2/0 myeloma cellline to generate hybridomas that secrete murine antibodies. Supernatantsfrom polyclonal and sub-cloned monoclonal hybridomas were screened forbinding to human and cyno TIGIT-overexpressing Expi293 cells and forbinding affinity for human and cyno TIGIT recombinant proteins usingstandard SPR methods. Murine antibodies from selected hybridomas werepurified and characterized extensively in binding and functional assays.Five functional and cyno cross-reactive murine antibodies were humanizedto contain a hIgG4 framework (inclusive of an optional hinge variant asoutlined herein) and isotype. The sequences are shown in FIG. 53.

L. Example 12: FACS KD Measurements of Phage and Hybridoma-DerivedAntibodies Binding to Cells Over-Expressing Human and Cyno TIGIT

1. Protocols

The following cell lines were prepared to estimate the bindingaffinities of human phage and mouse anti-TIGIT antibodies: Expi293Parental, Expi293 human TIGIT over-expressing, and Expi293 cyno TIGITover-expressing. The following hybridoma and phage antibodies were eachprepared in an 11-point 2-fold serial dilution series at a binding siteconcentration range of 195 pM-200 nM:

Phage generated antibodies: CPA.9.027, CPA.9.049, CPA.9.059.

Hybridoma generated antibodies (pre-humanization): CHA.9.536, CHA.9.541,CHA.9.543, CHA.9.546, CHA.9.547 and CHA.9.560. Included were twodifferent benchmark antibodies, BM26 (WO2016/028656A1, Clone 3106 asmouse IgG1) and BM29 (US2016/0176963A1, Clone 22G2 as mouse IgG1).

The 12th well of each titration contained buffer only to serve asbackground. Each cell type was incubated with an anti-human TIGIT mAbfor 60 minutes at 4° C. After washing, AF647-tagged goat anti-humanF(ab′) (Jackson Immunoresearch) and AF647-tagged goat anti-mouse IgG-Fc(Southern Biotech #1030-30) were added to cells incubated with human andmouse mAbs, respectively. A FACS Canto II HTS instrument then recordedthe Geometric Mean Fluorescence Intensity (gMFI) of 5000-10,000 eventsfor each well. A plot of the gMFI as a function of the human PVRmolecular concentration was fit using Graphpad Prism's “one site,specific binding” model to estimate the KD and the 95% confidenceintervals of each nonlinear fit.

2. Results

The two independent FACS KDs measured for each mAb differed by no morethan 2-fold on average. A single representative measurement for KD alongwith the 95% confidence interval of the binding isotherm fit is listedfor each mAb for human and cyno over-expressing cells in FIG. 54 andFIG. 55, respectively. CPA.9.059 did not show binding to the cynoover-expressing cells. It should be noted that the binding siteconcentrations (2× the molecular concentration) for all mAbs are usedfor the nonlinear curve-fitting, which means the assumption is made thatthis FACS KD method is measuring the binding site constant (kD) ratherthan the molecular or stoichiometric binding constant.

M. Example 13: FACS Blocking Assay of Phage and Hybridoma-DerivedAnti-Human Tigit Mabs Inhibiting PVR-FC Binding to Tigit

1. Introduction

The purpose of this assay is to characterize phage and hybridoma-derivedanti-human TIGIT antibodies' ability to inhibit the binding of human PVRto human TIGIT over-expressed on a cell surface. First, the humanTIGIT-human PVR binding affinity will be determined by FACS. The bindingisotherms showed the saturating concentration of human PVR which wasused for the blocking assays. Next, cells over-expressing human TIGITcells were titrated with phage and hybridoma-produced anti-TIGIT mAbs,followed by adding a saturating concentration of human PVR. Anti-humanTIGIT antibody binding on the over-expressing cells were then measuredusing FACS.

2. Protocols

FACS KD Assay:

Various human PVR-Fc isotypes were tested via FACS for optimal bindingand it was determined human PVR-h1Fc (Sino Biological #10109-H20H) andhuman PVR-m2aFc (Compugen) showed the highest binding levels to humanTIGIT over-expressing cells. The two PVR isotypes were each 2-foldserially diluted over an 11-point titration series at a final molecularconcentration range of 98 pM-100 nM. The 12th well of each titrationcontained buffer only to serve as background. Each cell type wasincubated with mAb for 60 minutes at 4° C. while. After washing,AF647-tagged F(ab′)2 fragment goat-anti human Fc (Jackson Immunoresearch#109-606-098) and AF647-tagged goat anti-mouse IgG (SouthernBiotech#1033-31) were added to wells titrated with human and mouse anti-TIGITmAbs, respectively. A FACS Canto II HTS instrument then recorded theGeometric Mean Fluorescence Intensity (gMFI) of 5000-10,000 events foreach well. A plot of the gMFI as a function of the human PVR molecularconcentration was fit using Graphpad Prism's “one site, specificbinding” model to estimate the KD and the 95% confidence intervals ofeach nonlinear fit. Results of human PVR-m2aFc and human PVR-h1Fc areshown in FIGS. 57A and B, respectively.

Phage MAbs Blocking Assay:

The following phage-derived hIgG4 antibodies and benchmark mAbs wereeach prepared in a three-point 5-fold serial dilution series at abinding site concentration range of 267 pM-6.7 nM: CPA.9.027, CPA.9.049and CPA.9.059, as well as BM26 (WO2016/028656A1, Clone 3106 as hIgG4)and Synagis hIgG4 (negative isotype control).

The 4th well of each titration contained buffer only to serve as abackground. Cells were incubated with mAb for 15 minutes at 4° C. HumanPVR-m2aFc (Compugen) was then incubated for 1 hour at 4° C. Afterwashing, AF647-tagged goat anti-mouse IgG (SouthernBiotech #1033-31) wasadded. A FACS Canto II HTS instrument then recorded the Geometric MeanFluorescence Intensity (gMFI) of 5000-10,000 events for each well. ThegMFI values of bound human PVR for the cells pre-incubated with the mAbswere compared to gMFI values of cells pre-incubated with the blockingbenchmark mAb and non-blocking control mAb. If a phage antibody reducedthe human PVR-m2aFc binding signal compared to the signal from thetitration with the known non-blocking mAb, the antibody wascharacterized as blocking PVR binding at that concentration of phagemAb. The blocking trends of the phage mAbs were similar to the PVRblocking with the BM26 benchmark (FIG. 58).

Hybridoma MAbs Blocking Assay:

The following hybridoma antibodies were each prepared in an 11-point2.5-fold dilution series at a binding site concentration range of 14pM-133 nM: CHA.9.536, CHA.9.541, CHA.9.546, CHA.9.547, CHA.9.560, BM26(WO2016/028656A1, Clone 3106 as mouse IgG1) and BM29 (US2016/0176963A1,Clone 22G2 as mouse IgG1).

The 12th well of each titration contained buffer only to serve asbackground. Cells were incubated with mAb for 15 minutes at 4° C. HumanPVR-hlFc (Sino Biological #10109-H20H) was then added, and the cellswere then incubated for 1 hour at 4° C. After washing, AF647-taggedF(ab′)2 fragment goat-anti human Fc (Jackson Immunoresearch) was added.A FACS Canto II HTS instrument then recorded the Geometric MeanFluorescence Intensity (gMFI) of 5000-10,000 events for each well. Aplot of the gMFI as a function of the mAb binding site concentration wasfit nonlinearly using Graphpad Prism's “log(inhibitor) vs.response—Variable slope (four parameters)” model to estimate the IC50 ofeach nonlinear fit. This experiment was repeated twice over two days.

3. Results

FIGS. 58 and 59 Demonstrate that Both the Phage and Hybridoma AntibodiesPotently Block the Binding of Human PVR-Fc to Human TIGIT Over-Expressedon the Cell-Surface of Expi293 Cells.

The blocking activity of the phage and hybridoma antibodies iscomparable to the two benchmark antibodies tested, BM26 and BM29.

N. Example 14: Surface Plasmon Resonance (SPR) Kinetics Studies of NinePhage- and Hybridoma-Derived Mabs Binding to Human, Cyno, and MouseTigit

1. Protocols

All experiments were performed using a ProteOn XPR 36 instrument at 22°C. First, high density capture surfaces were prepared with goat antihuman Fc polyclonal antibody (Thermo # H10500) and rabbit anti mouseantibodies (GE Healthcare # BR100838), respectively, immobilized overall vertical capture lanes and horizontal interspots on separate GLCchips using standard amine coupling. Typical immobilization levels forthe anti-human capture pAb and the anti-mouse capture antibody for eachGLC chip were around 5000 RU. Human TIGIT was obtained from SinoBiologicals while mouse TIGIT monomer and cyno TIGIT monomer wereprepared in-house. The purified mAbs studied for binding to human,mouse, and cyno TIGIT are listed below:

Phage antibodies: CPA.9.027, CPA.9.049 and CPA.9.059

Hybridoma antibodies: CHA.9.536, CHA.9.541, CHA.9.543, CHA.9.546,CHA.9.547 and CHA.9.560

Benchmark comparisons: BM26 (WO2016/028656A1, Clone 3106 as hIgG4) andBM29 (US2016/0176963A1, Clone 22G2 as hIgG4).

Each mAb was diluted to ˜0.5 μg/mL in running buffer which was 1×PBSTwith filtered BSA added to a final concentration of 100 μg/mL. For each“single-shot kinetics” cycle on the ProteOn instrument, a different mAbwas captured over one of the six unique vertical capture lanes forapproximately 1.5-2.5 minutes. After switching the buffer flow of theProteOn to the horizontal direction, capture surfaces were stabilizedfor approximately 15-20 minutes. Six concentrations of a 3-fold dilutionseries of human TIGIT (346 pM-84.1 nM), cyno TIGIT (371 pM-90.2 nM), ormouse TIGIT (382 pM-92.9 nM) were injected for 2 minutes followed by 20minutes of dissociation at a flow rate of 504/min. An identical bufferinjection preceded each series of injected antigen fordouble-referencing. Anti-human antibody surfaces were regenerated withtwo 30-second pulses of 146 mM phosphoric acid and anti-mouse antibodycapture surfaces were regenerated with two 30-second pulses of 10 mMglycine, pH 1.7. The sensorgrams of TIGIT antigen injected over capturedmAbs were processed using a ProteOn version of Scrubber and were fit toa 1:1 kinetic binding model including a term for mass transport.

FIG. 56 shows the resulting kinetic rate constants and the equilibriumdissociation constants where data were reliable enough to estimate thebinding constants (sensogram data not shown). The asterisks indicate thekd values that had to be held constant at 1.00×10−5/sec. In cases suchas clone CHA.9.560 binding to human TIGIT, the kinetic model was able toestimate a Kd, but it is it virtually impossible to accurately estimatea Kd on the order 1×10⁻⁶/sec after only 20 minutes of dissociation datagiven the sensitivity of the instrumentation.

O. Example 15: Functional Analyses of Anti-Tigit Antibodies

1. Rationale and Objectives

To functionally characterize the ability of anti-human TIGIT antibodiesto inhibit the interaction of TIGIT and its ligand PVR, and toconsequently enhance human T cell activation either as a monotherapy orin combination with an anti-human PVRIG antibody, CHA.7.518.1.H4(S241P).

2. Protocols

Human TIGIT/CD155 Jurkat IL-2 Luciferase Reporter Assay:

The human TIGIT/PVR Jurkat IL-2 luciferase reporter bioassay kit(Promega) was utilized to assess the effect of anti-human TIGIT antibodytreatment on T cell activation. Jurkat T cells were stably transfectedwith recombinant human TIGIT and a luciferase reporter gene driven bythe IL-2 response element (IL-2-RE). The stimulator cells wereartificial APC (aAPC) CHO-K1 cells expressing recombinant human PVR, andan engineered cell surface protein designed to activate TCR-mediatedsignaling in an antigen-independent manner. Following co-culture ofthese cells, the human TIGIT/human PVR interaction inhibits TCRsignaling and IL-2-RE-mediated luminescence. Addition of an anti-humanTIGIT antibody that blocks the human TIGIT/human PVR interactionreleases the inhibitory signal, resulting in T cell activation andIL-2-RE-mediated luminescence. The assay was carried out according tothe manufacturer's instructions. Briefly, aAPC CHO-K1 human PVR cellswere thawed in a 37° C. water bath and diluted in F-12 mediumsupplemented with 10% FBS (Promega). 25,000 cells/well were plated onwhite, flat-bottom tissue culture treated 96 well plates (Costar).Plates were then incubated overnight at 37° C. The next day, hybridomaand phage-derived anti-human TIGIT antibodies, mouse IgG1 (mIgG1) andhIgG4 isotype control antibodies, or benchmark (BM) anti-human TIGITantibodies were added either as a single dose at 10 μg/ml, or in a 10point, 2-fold dilution series starting at 20 μg/ml. Jurkat IL-2-REluciferase human TIGIT cells were thawed in a 37° C. water bath anddiluted in RPMI medium supplemented with 10% FBS (Promega). 125,000Jurkat cells were added to each well. Plates were then incubated at 37°C. with 5% CO2 for 6 hours. After the incubation, plates were removedfrom the incubator and allowed to equilibrate to room temperature for 30minutes. 80 μl of Bio-Glo luciferase substrate (Promega) was added toeach well and the mixture was allowed to equilibrate for 10 minutes atroom temperature protected from light. Luminesce was quantified on anEnVision multi-label reader (Perkin Elmer) with an ultra-sensitiveluminescence detector. Luminesce signal was reported in relative lightunits (RLU).

Human CMV-Specific CD8⁺ T Cell Expansion:

Human CMV-reactive peripheral blood mononuclear cells (PBMCs) (CTL) werethawed, resuspended at 2×10⁶ cells/ml, and stimulated with 1 μg/ml ofthe CMV pp65 peptide (Anaspec) in complete RPMI medium supplemented with2 ng/ml recombinant human IL-2 (R&D systems) and 10 ng/ml recombinanthuman IL-7 (R&D systems) at 37° C. with. After 9 days, cells were split1:2 and rested with low dose human IL-2 (100 IU/ml). The frequency ofCMV-specific CD8⁺ T cells was determined with the CMV pp65/HLA-A2tetramer (MBL). CMV-specific CD8⁺ T cells that were 65-98% tetramerpositive were utilized in assays between days 12 and 16 following CMVpeptide stimulation.

Human Cmv-Specific Cd8+ t Cell Co-Culture Assay with HumanPvr-Expressing Melanoma Cell Lines:

An in vitro co-culture assay with human CMV-specific CD8+ T cells wasutilized to assess the effect of anti-human TIGIT antibodies onantigen-specific cytokine secretion. The target cell line used in theassay was the HLA-A2⁺ melanoma cell line, Mel624 stably transduced witha lentivirus containing human PVR DNA (System Biosciences). A stablepool of Mel624 human PVR over-expressing cells were pulsed with the CMVpp65 peptide at 0.0033 μg/ml or 0.001 μg/ml at 37° C. for 1 hour. Cellswere then washed and plated at 50,000 cells/well. Hybridoma and phagederived anti-human TIGIT antibodies, control mIgG1 or hIgG4 isotypeantibodies, or BM anti-human TIGIT antibodies were added at aconcentration of 10 μg/ml. Human CMV-specific CD8⁺ T cells from threedifferent donors, specified as Donor 2, Donor 4, and Donor 210 wereexpanded according to the protocol above. 50,000 human CD8⁺ T cells wereadded to each well. Co-cultures were incubated at 37° C. with 5% CO2 for24 hours. After the incubation, plates were centrifuged at 1200 rpm for1 minute and the supernatant was collected. The amount of humaninterferon gamma (IFNγ) in the co-culture supernatant was measured byflow cytometry using a cytometric bead assay (BD).

Human CMV-Specific CD8⁺ T Cell Co-Culture Assay with Human PVR- andHuman PVRL2 (CD112)-Expressing Melanoma Cell Lines:

The combined effect of anti-human TIGIT antibodies andCHA.7.518.1.H4(S241P), an anti-human PVRIG antibody, on antigen-specificcytokine secretion was assessed by an in vitro co-culture assay withhuman CMV-specific CD8⁺ T cells similar to the assay described above.The target cell line used in the assay was the HLA-A2⁺ melanoma cellline, Mel624, which stably expressed human PVR and human PVRL2, theligands for TIGIT and PVRIG, respectively, through lentiviraltransduction (System Biosciences). The human PVR and human PVRL2overexpressing Mel624 cells were pulsed with the CMV pp65 peptide at0.0033 μg/ml or 0.001 μg/ml at 37° C. for 1 hour. Cells were then washedand plated at 50,000 cells/well. Hybridoma and phage derived anti-humanTIGIT antibodies, or a BM anti-human TIGIT antibody, were added to theculture in combination with CHA.7.518.1.H4(S241P) or a control hIgG4isotype antibody at 10 μg/ml. Human CMV-specific CD8⁺ T cells from threedifferent donors, specified as Donor 4, Donor 25, and Donor 210 wereexpanded, according to the protocol above. 50,000 human CD8⁺ T cellswere added to each well. Co-cultures were incubated at 37° C. for 24hours. After the incubation, plates were centrifuged at 1200 rpm for 1minute and the supernatant was collected. The amount of human interferongamma (IFNγ) in the co-culture supernatant was measured by flowcytometry using a cytometric bead assay (BD).

3. Results

Anti-Human TIGIT Antibodies Enhance IL-2 Signaling:

The ability of hybridoma and phage-derived anti-human TIGIT antibodiesto enhance IL-2 signaling was assessed with the human TIGIT/human PVRJurkat luciferase reporter assay. FIG. 60 and FIG. 62 demonstrate theeffect of 10 μg/ml phage or hybridoma-derived anti-human TIGITantibodies on IL-2 signaling, respectively. Three phage-derivedantibodies, CPA.9.027, CPA.9.049, and CPA.9.059 robustly enhanced IL-2signaling compared to the hIgG4 isotype control. In addition, all threephage antibodies induced more IL-2 signaling compared to the BManti-human TIGIT antibodies, BM26 and BM29. The five hybridoma-derivedantibodies, CHA.9.536, CHA.9.541, CHA.9.546, CHA.9.547 and CHA.9.560also induced IL-2 signaling compared to the mIgG1 isotype control. Ofnote, the five hybridoma antibodies induced similar IL-2 signalingcompared to BM26 and BM29. The anti-human TIGIT non-blocking antibody,CHA.9.543 did not significantly increase IL-2 signaling. To determinewhether the effect of anti-TIGIT antibodies was dose-dependent, theassay was carried out with a 10 point, 2-fold dilution series for eachantibody starting at 20 μg/ml (FIGS. 61 and 63). IL-2 signalingdecreased in a dose-dependent manner with all eight anti-human TIGITantibodies, as well as BM26 and BM29.

Anti-Human TIGIT Antibodies Increase IFNγ Secretion from HumanCMV-Specific CD8+ T Cells:

The ability of hybridoma and phage-derived anti-human TIGIT antibodiesto modulate IFNγ secretion was assessed with the CMV-specific Tcell/Mel624 co-culture assay. FIG. 64 shows the effect of the anti-humanTIGIT antibodies on IFNγ secretion. Three phage-derived antibodies,CPA.9.027, CPA.9.049, and CPA.9.059 enhanced IFNγ secretion compared tothe media alone and hIgG4 isotype control antibody. Additionally, fivehybridoma derived antibodies, CHA.9.536, CHA.9.541, CHA.9.546, CHA.9.547and CHA.9.560 also increased IFNγ production compared to the mIgG1isotype control antibody. The phage and hybridoma-derived TIGITantibodies induced IFNγ in a similar manner to BM26 and BM29. Asexpected, the anti-human TIGIT non-blocking antibody, CHA.9.543 did notsignificantly effect IFNγ secretion.

FIG. 65 shows the combined effect of the anti-human TIGIT antibodies andCHA.7.518.1.H4(S241P) on IFNγ secretion. Three phage-derived antibodies,CPA.9.027, CPA.9.049, and CPA.9.059, and five hybridoma derivedantibodies, CHA.9.536, CHA.9.541, CHA.9.546, CHA.9.547 and CHA.9.560,including BM26, all enhanced IFNγ secretion compared to their respectiveisotype control antibodies, when either treated alone, or in combinationwith CHA.7.518.1.H4(S241P). The anti-human TIGIT non-blocking antibody,CHA.9.543 resulted in less IFNγ secretion compared to other anti-humanTIGIT antibodies. The percent increase of IFNγ secretion in eachantibody over respective isotype control antibodies is summarized inFIG. 66. A syergistic effect is observed in the combined treatment ofanti-human TIGIT antibodies and CHA.7.518.1.H4(S241P).

4. Summary and Conclusions

Addition of anti-human TIGIT antibodies to the human TIGIT/human PVRJurkat reporter assay induced a robust, dose-dependent increase in IL-2signaling. Additionally, the anti-human TIGIT antibodies increased IFNγsecretion from human CMV-specific CD8+ T cells when co-cultured withMel624 human PVR cells. The secretion of IFNγ was further increased byanti-human TIGIT antibodies in combination with an anti-human PVRIGantibody. Taken together, these data demonstrate that the anti-humanTIGIT antibodies can block TIGIT-mediated suppression of human T cellactivation, and T cell activation is enhanced by co-blockade of bothTIGIT and PVRIG.

P. Example 16: Binning Analysis of Anti-Tigit Antibodies

1. Protocols

Experiments were performed by Wasatch Microfluidics Inc. (Salt LakeCity, Utah) using a Continuous Flow Microspotter (CFM) and an IBIS MX96SPR Imager (MX96 SPRi). The following anti-human TIGIT mAbs and humanPVR-Fc variants were each diluted to ˜10 μg/mL in 10 mM sodium acetate,pH 5.0 and covalently immobilized using standard amine coupling onindependent spots of a Xantec 200M biosensor prism chip for 7-minutecycles using the CFM:

1 CPA.9.009-H4 2 CPA.9.011-H4 3 CPA.9.012-H4 4 CPA.9.013-H4 5CPA.9.014-H4 6 CPA.9.015-H4 7 CPA.9.018-H4 8 CPA.9.027-H4 9 CPA.9.049-H410 CPA.9.053-H4 11 CPA.9.057-H4 12 CPA.9.059-H4 13 CPA.9.064-H4 14CPA.9.069-H4 15 CPA.9.071-H4 16 CPA.9.077-H4 17 CPA.9.081-H4 18CHA.9.519 19 CHA.9.521 20 CHA.9.522 21 CHA.9.527 22 CHA.9.528 23CHA.9.529 24 CHA.9.535 25 CHA.9.536 26 CHA.9.541 27 CHA.9.546 28CHA.9.547 29 CHA.9.549 30 CHA.9.552 31 CHA.9.554 32 CHA.9.555 33CHA.9.560 34 CHA.9.525 35 CHA.9.538 36 CHA.9.543 37 CHA.9.553 38CHA.9.556 39 CHA.9.561 40 BM8-H4 41 BM9-H4 42 BM26-H4 43 BM29-H4 44MBSA43-M1 45 PVR-Fc M2A 46 Sino PVR-Fc

BM8-H4 and BM9-H4 refer to (US2015/0216970A1, Clones 10A7 and 1F4reformatted as hIgG4), respectively. MBSA43-M1 is a mouse anti-humanTIGIT IgG1 from eBioscience. The prism chip was then rinsed with 1×PBSTfor 3 minutes and then directly loaded into the MX96 SPRi imager whereexcess NHS esters were quenched with a 5-minute injection of 1 Methanolamine. Preliminary experiments included several cycles ofinjecting 100 nM monomeric human TIGIT (Sino Biologicals, Cat#10917-H08H) over all immobilized mAbs for four minutes followed byregeneration in order to test the binding activity of the antibodies andto best determine the regeneration conditions by assessingreproducibility of the TIGIT binding. These preliminary experimentsshowed that the best reagent to reproducibly regenerate most of theimmobilized mAbs was a 30-second pulse of 1/500 phosphoric acid. Theimmobilized PVR, however, did not retain activity and therefore theirblocking patterns were generated and “binned” as analytes in solutiononly. In these preliminary experiments and the binning experimentsdescribed below, all protein samples were prepared in the running bufferwhich was degassed HBST. A “sandwich” epitope binning protocol wasperformed where each mAb and PVR was injected over TIGIT pre-complexedto each immobilized mAb to determine whether or not the immobilized mAbblocks the mAb in solution from binding to TIGIT. For each cycle 100 nMof TIGIT was first injected over all immobilized mAbs for 4 minutesfollowed immediately by a 4-minute injection of a competitor mAb orligand at 274 nM (binding site concentration). This was repeated witheach mAb and PVR acting as the competitor analyte. Control cycles withrunning buffer instead of competitor protein were performed after every12 cycles for double-referencing. All surfaces were regenerated aftereach cycle with a 30 second pulse of 1/500 phosphoric acid. Sensorgramdata were processed and referenced using Wasatch's proprietary software.An antibody pair was classified as having a shared TIGIT-binding epitopeif no binding was observed from the injection of competitor over TIGITpre-complexed to immobilized mAb. An antibody pair was classified asbinding to different epitopes on TIGIT, or “sandwiching”, if theinjection of competitor mAb showed binding to the pre-complexed TIGIT.Low or minimal binding of competitor was classified as an “intermediate”blocker. Hierarchical clustering of the pair-wise TIGIT blockingpatterns for each mAb and ligand was performed using Wasatch'sproprietary software.

2. Results

Both PVR-Fc proteins and 13 of the mAbs either lost activity or couldnot be regenerated as ligands so their blocking patterns were determinedas analytes in solution only. MAb CPA.9.014-H4 was not binned because itshowed no binding to TIGIT. FIG. 67 shows the dendrogram clusteringbased on the pair-wise blocking patterns for each mAb and two PVRproteins. The vertical axis represents the statistical similarity factorin the blocking patterns. Wasatch Microfluidics applied a cut-off factorof 5 to cluster the mAbs which is indicated by the line in FIG. 67. Forthe strictest definition of an epitope “bin” where only those mAbs (andPVRs) which show identical blocking patterns bin together, there are atotal of 12 discrete bins. If blocking patterns that show only minimaldifferences are clustered together, there are four closely related“communities” of mAbs and PVRs. These “communities” are indicated withdifferent shaded blocks on the bottom of FIG. 67. FIG. 68 groupstogether the mAbs and PVRs that populate each discrete, unique bin witheach bin indicated by a black box. Gray boxes surround all the uniquebins that make up each “community” of related blocking patterns. ThemAbs and PVRs in FIG. 68 are listed with the number key which representseach protein in the dendrogram in FIG. 67.

Q. Example 17: Administration of Anti-PVRIG Antibodies to Tigit KnockOut Mice

Rationale and Objectives

To examine whether TIGIT deletion in combination with mouse PVRIGblockade can enhance tumor growth inhibition and survival in a syngeneicmouse tumor model.

Protocols

Animals

TIGIT knockout (KO) mice were generated at Ozgene Pty LTD (Australia).

C57BL/6 wild type (WT) mice (Ozgene) served as controls. Eight to elevenweeks old female TIGIT KO and C57BL/6 mice were used. All studies wereapproved by the Institutional Animal Care and Use Committee at theTel-Aviv University (Tel-Aviv, Israel).

In Vivo Tumor Models

1×105 B16/Db-hmgp100 melanoma cells were inoculated subcutaneously(s.c.) into the right flank of C57BL/6 WT or TIGIT KO mice. Antibodytreatment was initiated on the same day as tumor inoculation (day 0),with 7-10 mice per treatment group. Antibodies used were the mouse IgG1isotype control (Clone MOPC-21 BioXcell), and mouse IgG1 anti-mousePVRIG (Clone 407, Compugen LTD). Antibodies were administrated at 10mg/kg by intra-peritoneal injection, twice per week for 3 weeks. Tumorgrowth was measured with electronic caliper every 2-3 days and wasreported as 0.5×W2×L mm3 (L is length and W is width of the tumor).Animals reaching 2250 mm3 tumor size were anesthetized.

Statistical Analysis

Two-way ANOVA with repeated measures, followed by two-way ANOVA withrepeated measures for selected pairs of groups was performed using JUMPsoftware (Statistical Discoveries TM). Analyses of tumor growthmeasurements were performed by comparing tumor volumes measured on thelast day on which all study animals were alive. Statistical differencesin percentage of mice tumor free were determined by a Log RankMantel-Cox test. Values of P<0.05 were considered significant. * p<0.05;** p<0.01; *** p<0.001.

Results

In vivo tumor growth inhibition following treatment with anti-mousePVRIG blocking antibody in TIGIT KO mice We tested the in vivo efficacyof TIGIT deletion in combination with mouse PVRIG blockade in asyngeneic mouse B16/Db-hmgp100 subcutaneous melanoma tumor model.Treating tumor bearing C57BL/6 WT mice with an anti-mouse PVRIG blockingantibody had a minor effect on tumor growth inhibition (TGI) compared tothe isotype treatment (17% TGI at day 11 and 8% TGI at endpoint, day18). The effect of TIGIT deletion on tumor growth was minor compared toC57BL/6 WT control group (17% TGI at day 11 and 13% TGI at endpoint).However, when TIGIT deletion was combined with anti-mouse PVRIG antibody(Clone 407) treatment, significant TGI was evident (63% at day 11 and49% TGI at endpoint) (FIGS. 80A and 80B). In accordance to TGI, TIGIT KOmice treated with the anti-mouse PVRIG antibody (Clone 407) exhibitedincreased survival compared to the C57BL/6 WT control group, however,statistical significance was not achieved (FIG. 80C).

Summary and Conclusions

The combination of TIGIT deletion and PVRIG blockade significantlyreduced tumor growth in vivo, indicating that both TIGIT and PVRIG playan inhibitory role in this melanoma tumor model. These data suggest thatco-targeting TIGIT and PVRIG could be another combination therapy thatsignificantly enhances anti-tumor responses.

R. EXAMPLE 18: PVRIG ANTAGONISM ENHANCES T CELL EFFECTOR

FUNCTION AND REDUCES TUMOR GROWTH

Abstract

Despite recent advances, the majority of patients do not derive longterm benefit from checkpoint inhibitors. PVRIG is a novel immunesuppressive receptor of the DNAM/TIGIT family and we demonstrate here arole of PVRIG in regulating anti-tumor responses. PVRIG binds to PVRL2and displays significantly enhanced expression on tumor infiltratinglymphocytes as compared to lymphocytes from normal tissues. PVRIGantagonism enhanced human T cell activation and combination of PVRIGwith either PD-1 or TIGIT inhibitors further synergistically increasedlymphocyte function. We next addressed the role fo PVRIG in preclinicaltumor models. PVRIG^(−/−) mice displayed significantly increased T cellactivation in vitro and reduced MC38 tumor growth that was mediated byincreased CD8 effector function. Antagonistic anti-PVRIG antibodysignificantly reduced tumor growth in combination with anti-PD-L1 orwhen tested in TIGIT^(−/−) mice. In summary, we demonstrate thatPVRIG-PVRL2 pathway was induced in human cancers and that antagonizingPVRIG-PVRL2 interactions resulted in increased T cell function andreduced tumor growth.

State of Significance

These data demonstrate that PVRIG is a promising target for thetreatment of cancer and provide the rationale for testing a PVRIGinhibitor, CHA.7.518.1.H4(S241P), as a novel cancer immunotherapy agenteither as monotherapy or in combination with either TIGIT or PD1blockade.

Introduction

Increasing evidence demonstrate that endogenous immune responses arecritical in sculpting the initiation, progression, and suppression ofcancer (1) (2). The immune status of patients as well as the content oftumor-infiltrating leukocytes (TILs) within the tumor microenvironment(TME) are key prognostic indicators of not only cancer survival rates,but also how patients respond to therapy (3) (4). T cells are a keycomponent of TILs that can invoke an anti-tumor response, and mostanti-tumor immune responses ultimately rely on the functionality ofeffector lymphocytes cells. An enrichment of CD8 T cells in the TME of apatient's tumor, as well as other factors that bias an immune responsetowards an effective CD8 T cell response such as mutational load and aTh1 polarized TME, are all key prognostic indicators for a favorableanti-tumor immune response (5) (6).

A key observation across many solid tumors is that effector T cells havean activated or ‘exhausted’ phenotype within the TME (7). This indicatesthat although T cells within the TME have initially seen cognateantigen, been activated, and trafficked to the tumor, they aresubsequently not capable of invoking an effective anti-tumor response.Pre-activated or exhausted T cells are defined by increased surfaceexpression of co-inhibitory receptors, such as PD-1 and CTLA-4 (8).Therapeutically targeting these co-inhibitory receptors with antibodiesthat inhibit interactions with their cognate ligands have shownremarkable clinical efficacy in patients with multiple advanced cancers(9). Mechanistically, it has been shown that targeting theseco-inhibitory receptors leads to the expansion of already tumor-reactiveT cells that pre-exist in the TME and to the production of T cell poolswith widened T cell receptor diversity (10) (11) (12). Althoughcheckpoint inhibitors currently in the clinic have revolutionized cancertreatment and demonstrated the power of the immune system in combatingcancer, many patients still relapse and/or do not respond to treatment.Consequently, increased understanding of the immune response in cancerand targeting additional immune-based pathways will lead to additionaltherapeutic treatments.

Among these novel pathways, a group of receptors and ligands within thenectin and nectin-like family are currently under investigation aspotential novel cancer immunotherapies. Receptors within this familyinclude DNAM-1 (CD226), CD96 (TACTILE), TIGIT, and more recently, PVRIG(CD112R) (13) (14) (15). Of these molecules, DNAM is an activatingreceptor within this subfamily, binding to 2 ligands, PVR (CD155) andPVRL2 (CD112), to deliver an activating signal to lymphocytes (16). Tworeceptors in this family have been shown to inhibit human lymphocytefunction, TIGIT, and more recently, PVRIG (17) (18). TIGIT is reportedto have a high affinity interaction with PVR, a much weaker affinity toPVRL2, and has been shown to inhibit both T cell and NK cell responsesby delivering an inhibitory signal into lymphocytes through its ITSMmotif (19) (20). More recently, PVRIG was shown to bind with highaffinity to PVRL2 and to deliver an inhibitory signal through its ITIMmotif (15). In both cases, the affinity of TIGIT to PVR and of PVRIG toPVRL2 is higher than the affinity of DNAM to either PVR or PVRL2,suggesting TIGIT and PVRIG can outcompete PVR and PVRL2 from DNAM,providing an indirect mechanism by which TIGIT and PVRIG can reduce Tcell function. Within this family, PVR is also a ligand for CD96. Thefunction of CD96 has been reported to be inhibitory on mouse lymphocytes(21) but activating on human lymphocytes (22). Based on these data, wepostulate on human lymphocytes that 2 receptors, TIGIT and PVRIG, bindwith high affinity to PVR and PVRL2, respectively, to deliver inhibitorysignals to dampen T cell function.

Although human PVRIG has been shown to inhibit T cells response in onerecent report, the role of PVRIG and PVRL2 in cancer immune surveillanceis not well understood. In particular, the expression profile of thispathway in cancers and the role of PVRIG in regulating CD8 T cellanti-tumor responses has not been reported. Furthermore, functionalcharacterization of the mouse PVRIG gene and the effect of disruptingPVRIG-PVRL2 interaction in vivo in pre-clinical tumor models has notbeen reported. Herein, we elucidated the role of PVRIG in a cancersetting by reporting on PVRIG and PVRL2 expression profile in cancer andthe effect of PVRIG antagonism in tumor cell co-culture assays and inpreclinical tumor models. We demonstrate that PVRIG has a differentiatedexpression profile on T cell subsets compared to TIGIT or CD96 and thatPVRIG and PVRL2 expression were induced in cancer compared to normaladjacent tissues. In multiple human in vitro assay systems, ahigh-affinity PVRIG antagonistic monoclonal antibody(CHA.7.518.1.H4(S241P)) enhanced T cell function, in particular whencombined with anti-TIGIT or anti-PD1 antibody. In addition, we reportthe novel characterization of mouse PVRIG using antagonistic antibodiesor PVRIG deficient mice and demonstrate that inhibition of PVRIG-PVRL2interaction reduced tumor growth, with most potent effects incombination with PD-1 inhibition or TIGIT genetic deficiency.Collectively, this data shows that PVRIG is a critical inhibitoryreceptor in regulating T cell anti-tumor responses and support thedevelopment of CHA.7.518.1.H4(S241P), for clinical testing in cancerpatients.

Materials and Methods

Human Peripheral Blood and Tumor Expression Studies

Healthy donor human PBMCs were obtained from Stanford University inaccordance with the Declaration of Helsinki. Human tissues were providedby the Cooperative Human Tissue Network, a National Cancer institutesupported resource. Human cancer tissue and matched normal adjacenttissues were dissociated into single cells as per manufacturer'sprotocol (Miltenyi Biotec), Dissociated cells were analyzed by flowcytometry for expression of various targets on different cell subsets.For each target expression on an individual cell subset, a foldexpression value was calculated by taking the MFI value of targetdivided by the MFI value of the isotype control. Other investigators mayhave received samples from these same tissue specimens. The tumor typewas determined based on reviewing the pathology report for each sample.For IHC studies, anti-PVRL2 antibody (HPA-012759, Sigma) and PD-L1(Spi42, SpringBio) were used to stain tumor micro-arrays (Biochaininstitute) using conditions as described in the supplemental methods.Scoring was performed by 2 independent reviewers on duplicate cores fromthe same tumor.

PVRIG Antibody Generation and Characterization

Anti-human PVRIG and anti-mouse PVRIG antibodies were generated asdetailed in the supplemental methods. Briefly, antibody bindingspecificity and affinity were assessed by selective binding to PVRIGengineered cells with no detectable binding to cells that do no expressthe gene. Antagonistic activity of these anti-PVRIG antibodies wasdetermined using ELISA and FACS based assays in which the interaction ofPVRIG with PVRL2 was disrupted. For characterization in cell basedassays, antibodies were tested in several T cell-target cell co-cultureassay systems consisting of target cells that express PVRL2 in culturewith PBMC or tumor-derived T cells. gp100 specific T cells lines wereexpanded from melanoma tumors as previously described (23). CMVpp65reactive T cells were expanded from healthy donor PBMCs (CM immunospot)with CMVpp65 (495-503), IL-2, and IL-7 for 10 days. For combinationstudies, antibodies to PD-1, TIGIT, and PVRIG were used at 10 μg/ml.Cytokine concentrations in conditioned media was determined usingCytometric Bead Array (CBA) and FACS staining was performed as describedin the supplemental methods.

Characterization of Mouse PVRIG Expression and Function

Binding interactions of mouse PVRIG with mPVRL2 and mPVR were assessedby SPR and ELISA using recombinant PVRIG, PVRL2, and PVR proteins and byFACS using ectopically engineered PVRIG and PVRL2 overexpressing celllines or PVR or PVRL2 siRNA transfected cell lines. PVRIG and TIGITdeficient mice were generated as described in the supplemental methods.Expression analysis was performed to examine expression of PVRIG inspleen, lymph node, and tumor in various cell subsets. Cell functionalassays demonstrating a T cell modulatory activity for mouse PVRIG wereestablished using WT and PVRIG^(−/−) T cells and PVRL2 Fc or PVRL2ectopically expressed target cells as detailed in the supplementalmaterials and methods. CT26, MC38, and B16/Db-hmgp100 tumor models wereperformed as described in the supplemental methods. All studies wereapproved by the Institutional Animal Care and Use committee at theTel-Aviv University (Tel-aviv, Israel) or Johns Hopkins University(Baltimore, USA).

Results

PVRIG Expression is Highest on Effector T Cells of Peripheral Blood andTumors

The Ig superfamily (IgSF) consists of hundreds of proteins but only afew of them are T cell inhibitory receptors. Proteins of the IgSF tendto evolve quickly (24) and therefore sequence similarity among theseproteins is generally low and is not optimal for identifying novelimmune receptors. To identify novel immune checkpoints, we developedbioinformatic algorithms based on shared genomic and proteomiccharacteristics among known immune checkpoints, such as gene structure,protein domains, predicted cellular localization and expression pattern.Using these algorithms, PVRIG was identified as a novel immune receptor.A report has recently also demonstrated that human PVRIG (CD112R) bindsto PVRL2 and inhibits T cell function (15). However, the relevance ofthis pathway in regulating tumor immune surveillance has not beenreported. Here, we have elucidated the expression and function of PVRIGand PVRL2 in human cancers and preclinical tumor models. In peripheralblood from healthy donors, PVRIG was expressed exclusively onlymphocytes, with highest expression on CD8 T cells and NK cells (FIG.83A). Further subset analysis of T cells showed highest PVRIG expressionon CD8 or CD4 memory/effector T cell subsets in comparison with Tregsubset (FIG. 83B, FIG. 90A). The predominantly memory T-cell expressionpattern differentiates PVRIG from other receptors in the family (TIGIT,CD96) which tend to have equal or higher expression on Tregs compared tomemory/effector T cells. We further compared the expression kinetics ofPVRIG and TIGIT post T cell activation in 2 assay systems (CMV recallresponse FIG. 83C, DC-MLR FIG. 83D, FIG. 90B) and show that PVRIG hasdelayed kinetics of induction and more sustained expression at the latetimepoint as compared to TIGIT. The preferential expression of PVRIG onmemory/effector cells as compared to TIGIT suggests a unique role forPVRIG in regulating T cell responses.

The delayed and sustained induction of PVRIG expression on T cells afteractivation suggested that it could be expressed in the tumormicroenvironment. Next, we analyzed the expression of PVRIG onleukocytes from dissociated human tumors directly ex vivo by FACS.Expression of PVRIG was detected on CD8 T cells, CD4 T cells, and NKcells from multiple tumor types (FIG. 83E-G, FIG. 90C). PVRIG wasco-expressed with PD-1 and TIGIT on CD4 and CD8 T cells (FIG. 83F). Onaverage, higher expression was detected on CD4⁺ and CD8⁺ TILs frombreast, endometrial, head and neck, lung, kidney, and ovarian tumors ascompared to bladder, colorectal, and prostate. In tumor samples in whichPVRIG expression was low/not present ex vivo, activation with anti-CD3and anti-CD28 enhanced the expression of PVRIG, suggesting that TILexpression of PVRIG can be further induced upon re-activation (FIG.90D). For colon, lung, kidney, endometrial, and ovarian tumors, we wereable to obtain normal adjacent tissue from the same patient and performa comparison of PVRIG expression on lymphocytes isolated from the tumorvs normal tissue. TILS showed a significant induction of PVRIG on CD4and CD8 T cells as compared to cells isolated from matching normaladjacent tissues (NAT) (FIG. 90E). As with PBMCs, we further comparedPVRIG, TIGIT, and PD1 expression on Tregs vs CD8 T cells from lung,endometrial, and kidney tumors. On TILS, TIGIT expression was higher onTregs compared to CD8 T cells whereas for PVRIG and PD1, similar orhigher expression was observed on CD8 T cells compared to Tregs (FIG.83H). Next, we examined the co-regulation of PVRIG, TIGIT, and PD-1 on Tcell populations by correlation analysis of either the magnitude ofexpression on TILS ex vivo or the magnitude of the fold change inexpression between tumor and NAT. In both analyses, CD4 and CD8 T cellsdisplayed a positive and significant correlation between PVRIG and PD1or TIGIT on (FIG. 90F). Taken together, these data demonstrate thatPVRIG is expressed on T cells and NK cells from multiple human cancers,placing PVRIG as a novel inhibitory receptor target that may be criticalin regulating T cell function in the tumor.

PVRL2 Expression is Enhanced in Tumors Tissue Compared to NormalAdjacent Tissue

As PD-L1 expression has been demonstrated to help predict responses toPD-1 inhibitors, we examined whether the expression of PVRL2 wasconcomitant with expression of its cognate receptor, PVRIG, in humancancer tissues. Using an anti-PVRL2 antibody that we validated forstaining FFPE samples (FIG. 91A), we stained tumor microarrays (TMA)composed of lung, colon, skin, breast, ovarian/endometrial, and kidneycancer tissues and scored each core based on prevalence and intensity ofPVRL2 expression. PVRL2 expression was not present or minimallyexpressed in the majority of normal tissue samples from these organs. Intumor tissues, PVRL2 expression on tumor cells was detected in ˜50-70%of lung, colon, breast, and ovarian/endometrial cancers (FIG. 84A, 84F).Expression in kidney cancer samples ranged from 20-40% whereasexpression in melanoma was the lowest (˜10%) (FIG. 84A, 84F). PVRL2expression was detected on tumor cells and immune cells at the invasivefront (FIG. 84B). To determine the specific immune cell subsetsexpressing PVRL2, we performed flow cytometry on freshly dissociatedtumors. Expression of PVRL2 was detected on CD45⁺ immune cells,particularly myeloid cells (e.g. CD14⁺ tumor associated macrophages(TAMs) and myeloid DCs) and on CD45⁻ non-immune cells from multipletumor types (FIG. 84C, D). No expression of PVRL2 was detected onlymphocytes (data not shown). Comparison of PVRL2 expression on CD45⁻cells and TAMs isolated from colon, lung, kidney, endometrial, andovarian tumors showed a significant induction of PVRL2 on cells isolatedfrom the tumor as compared to cells isolated from matching NAT of thesame donor (FIG. 92). For samples where we obtained PVRIG and PVRL2expression, we examined expression of PVRIG on lymphocytes compared withPVRL2 on myeloid and on CD45⁻ cells from multiple tumor types. Of thecancer types examined, endometrial, lung, and kidney cancers had thehighest prevalence of PVRIG^(hi) lymphocytes and PVRL2^(hi) TAMs orCD45⁻ non-immune cells (FIG. 842E, FIG. 93). Integrating data the TMAand dissociated tumor studies, we demonstrate that breast, endometrial,lung, head and neck, kidney, and ovarian tumors may representative aresponsive tumor type for PVRIG antagonism.

Compared to PD-L1, PVRL2 Expression is Differentially Regulated andPresent in PD-L1⁻ Tumors

As PVRIG and PD-1 can be co-expressed on tumor-infiltrating lymphocytes(TILs), we also examined the co-expression of PVRL2 and PD-L1 on thesame tumor by staining serial sections of the same TMA. PVRL2 expressionon tumor cells was clearly detected in PD-L1⁻ tumor samples (as definedby no membranous PD-L1 staining on tumor cells or immune cells) atsimilar frequency and average score compared to PD-L1⁺ samples. (FIG.85A, FIG. 84F). On immune cells, 3 of 5 tumors in which PVRL2 expressionwas detected on immune cells also expressed PD-L1 (data not shown), butthe small numbers of samples makes it difficult to conclude on immunecell co-expression of PD-L1 and PVRL2. The expression of PVRL2 on tumorcells in PD-L1 negative tumors suggested that PVRL2 expression was moreprevalent than PD-L1 in some tumors types and that targeting thispathway may be particularly effective in PD-L1⁻ tumors. Whereas PD-L1 isinduced primarily by IFN-γ as a mechanism of adaptive resistance (28),PVRL2 is modulated by genomic stress, DNA damage, and tumor suppressorgenes (29,30). To further understand the distinct regulation of PD-L1and PVR/PVRL2, we next assessed the regulation of PVR, PVRL2 and PD-L1expression in tumor cell lines and in monocyte-derived DCs by exposureto various inflammatory stimuli (FIG. 85D). Treatment of DCs withpro-inflammatory signals generally lead to an increase in PVR, PVRL2,and PD-L1 expression, demonstrating that PVR, PVRL2, and PD-L1expression are increased upon DC maturation. In contrast, treatment ofepithelial cells with IFN-γ increased expression of PD-L1 but had noeffect on the high baseline expression of PVRL2 (FIG. 85E), supportingdifferential regulation of PVRL2 expression in comparison with PD-L1 byIFN-γ. In summary, these findings indicate that PD-L1 and PVRL2 can beco-regulated on antigen presenting cells (APCs) such as DCs but can bedifferentially regulated on epithelial cells. The presence of PVRL2 inPD-L1-negative tumors suggests that targeting this pathway may be ofpotential benefit in patients that are non-responsive to or progress onPD-1 inhibitors.

CHA.7.518.1.H4(S241P) is a High Affinity Humanized Monoclonal Antibodyto PVRIG that Disrupts the Interaction of PVRIG to PVRL2

To examine the functional consequences of antagonizing human PVRIG-PVRL2interactions, we generated a high affinity, antagonistic anti-PVRIGantibody, CHA.7.518.1.H4(S241P), which blocks the interaction of PVRIGand PVRL2. This antibody selectively bound HEK293 cells ectopicallyexpressing human PVRIG or cynomolgus macaque PVRIG and also bound Jurkatcells that endogenously express PVRIG with sub-nanomolar affinity (FIG.86A). In biochemical assays, CHA.7.518.1.H4(S241P) blocked theinteraction of PVRIG Fc with PVRL2⁺ HEK293 cells (FIG. 86B) and alsoblocked PVRL2 Fc binding to PVRIG⁺ HEK293 cells (FIG. 86C). Using thisantibody, we observed a functional effect of an antagonistic anti-PVRIGin several T cell assays. Artificial antigen-presenting cells (aAPC)ectopically expressing a cell surface anti-CD3 antibody and human PVRL2were generated and co-cultured with primary human CD4 T cells, either inthe presence of anti-PVRIG (CHA.7.518.1.H4(S241P)) or isotype control.PVRIG expression was induced on proliferating CD4 T cells uponco-culture with the CHO anti-CD3 aAPC (FIG. 94A). Antagonism of PVRIGwith CHA.7.518.1.H4(S241P) enhanced proliferation of CD4 T cells frommultiple donors (FIG. 86D). We also tested the effect of anti-PVRIG on 2human gp100 reactive CD8 T cell lines that were derived from melanomatumors. These T cell lines were individually co-cultured with aAPCsexpressing HLA-A2 and PVRL2 (FIG. 94B) in the presence of isotypecontrol IgG or anti-PVRIG antibodies. As observed in both lines,anti-PVRIG increased IFN-γ and TNF-α production by ˜20-50%. In a doseresponse assessment, CHA.7.518.1.H4(S241P) displayed single digitnano-molar EC50 values in multiple assays (FIG. 94C, D). These datacollectively demonstrate that antagonizing PVRIG-PVRL2 interactions withCHA.7.518.1.H4(S241P) resulted in increased T cell activation.

CHA.7.518.1.H4(S241P) in Combination with TIGIT or PD-1 InhibitorsResulted in Synergistic Enhancement of T Cell Function.

Combination of PVRIG and TIGIT blockade synergistically increased CD4 Tcell function in a T cell-dendritic cell co-culture assay (15),suggesting a role for this pathway in regulating T cell-APCinteractions. The effects of PVRIG and TIGIT blockade on CD8 T cells ina tumor cell co-culture setting has not been reported. As our tumorexpression profiling demonstrated expression of PVRL2 on CD45⁻ immunecells, we further explored the effect of targeting this pathway in Tcell—tumor cell co-cultures using 2 T cell assay systems. We firstperformed a co-culture of 2 gp100 tumor antigen specific CD8 T celllines with a melanoma cell line, MEL624, in the presence of anti-PVRIG,anti-TIGIT, or isotype control antibodies, either individually or incombination. MEL624 cells express both PVR and PVLR2 and both TIL-209and TIL-463 expressed PVRIG, TIGIT, and PD-1 (FIG. 86F). On TIL-209, weobserved that anti-PVRIG or anti-TIGIT alone did not increase IFN-γ andthat the combination of anti-PVRIG and anti-TIGIT synergisticallyincreased IFN-γ production (FIG. 86G). On TIL-463, we observed thatanti-PVRIG or anti-TIGIT modestly increased IFN-γ production, and thatcombination of anti-PVRIG and anti-TIGIT additively increased IFN-γ(FIG. 86G). In an additional assay system, we utilized CMVpp65-reactiveCD8 T cells as a model system to study human T cell responses. HLA-A2⁺CMVpp65 CD8 T cells were expanded in the presence of CMVpp65 (495-503)and expression of PVRIG, TIGIT, and PD-1 was observed on day 10 (FIG.86F). PVRIG was expressed on CMVpp65 specific CD8 T cells at similarmagnitude to what was observed in human cancer samples (FIG. 83). Astarget cells, we identified a PD-L1^(hi) (Panc05.04) and a PD-L1^(lo)(Colo205) HLA-A2⁺ cancer cell line that both expressed similar amountsof PVR and PVRL2 (FIG. 86F). We next performed a co-culture of theCMVpp65 reactive T cells with HLA-A2⁺ tumor cell lines pulsed with pp65(495-503) peptide in the presence of blocking antibodies to PVRIG,TIGIT, and/or PD-1. We observed that anti-PVRIG Ab increased IFN-γ by˜50% in the co-culture with Panc05.04 cells and minimally in theco-culture with Colo205 (FIG. 86I). Combination of anti-TIGIT withanti-PVRIG Ab synergistically increased IFN-γ production on both targetcell lines, resulting in a greater increase in IFN-γ compared to PD-1antibody alone (FIG. 86H). Combination of anti-PVRIG and anti-PD-1 alsoled to synergistic increases in IFN-γ production as compared toindividual antibody (FIG. 86I). Taken together, these data suggest apotent synergy of combining PVRIG and TIGIT or PVRIG and PD1 blockade inincreasing activation of human CD8 T cells upon interaction with tumorcells.

PVRIG Deficiency Resulted in Increased T Cell Proliferation and ReducedTumor Growth

Although the sequence for mouse PVRIG and its interaction with mousePVRL2 has been reported, the expression profile and immune modulatoryactivity of mouse PVRIG is not well understood. We first analyzed mPVRIGRNA expression and transcript in NK, NKT and T cells (FIG. 87A).Activated mouse CD8 T cells had elevated PVRIG transcripts with delayedinduction kinetics compared to TIGIT (FIG. 87B). We confirmed that thatrecombinant mPVRIG bound to mPVRL2 protein by surface plasmon resonance(SPR) and ELISA performed in several assay orientations (FIG. 95A-D). Wealso observed an interaction between mPVRIG and mPVR, although theaffinity was approximately 10× less than the interaction with mPVRL2(FIG. 95E). To determine whether PVR or PVRL2 is the dominant ligand formPVRIG, we tested the binding of mouse PVRIG Fc to B16F10 cells whichexpress PVR and PVRL2 (data not shown). PVRIG Fc showed a dose dependentbinding to B16F10 cells that was completely abolished upon PVRL2 siRNAknockdown in B16F10 cells (FIG. 95F). In comparison, the binding ofPVRIG Fc fusion protein was slightly, but consistently, reducedfollowing PVR knockdown (FIG. 95F) suggesting that a very weakinteraction occurs between mPVRIG and mPVR. Taken together, theseresults demonstrate that in mice, PVRL2 is the primary ligand for PVRIG,as is the case in human. To delineate the role of PVRIG in immuneresponses, we generated PVRIG deficient (^(−/−)) mice (FIG. 96),PVRIG^(−/−) mice were born at the expected Mendelian ratios, displayedno overt phenotype up to 10 months of age, and at 8 weeks of age hadsimilar leukocyte cellularity (peripheral and lymphoid tissue) whencompared to wild type mice (FIG. 97). Wild-type (WT) CD8 T cells and NKcells express PVRIG and no expression of PVRIG was detected onPVRIG^(−/−) cells (FIG. 87C). To examine the role of PVRIG in regulatingmouse T cell responses, we examined the proliferation of WT andPVRIG^(−/−) T cells in 2 assay systems. WT or PVRIG^(−/−) T cells wereactivated with immobilized anti-CD3 in the presence of soluble PVRL2 Fcor control Fc protein. Soluble PVRL2 Fc significantly inhibited WT CD4⁺T cell proliferation but not PVRIG^(−/−) CD4⁺ T cell proliferation (FIG.87D), suggesting that PVRIG^(−/−) cells lack an inhibitory signal. Toevaluate the role of mouse PVRIG in CD8⁺ T cell interaction with tumorcells, PVRIG^(−/−) mice were bred to pmel TCR transgenic mice, whichexpress a transgenic TCR specific to gp100₂₅₋₃₃ (28). ActivatedPVRIG^(−/−) or WT Pmel CD8+ T cells were co-cultured with B16-Db/gp100melanoma tumor cells that endogenously express PVRL2 (data not shown)and activation and effector function evaluated. PVRIG^(−/−) pmel CD8⁺ Tcells showed enhanced degranulation and production of effector cytokines(IFN-γ and TNF-α) compared to WT cells (FIG. 87E). These data indicatethat mouse PVRIG inhibits activation and effector function oftumor-specific T cells upon co-culture with PVRL2⁺ tumor target cells.

We next studied the effects of PVRIG deficiency on tumor growth in theMC38 syngeneic model. PVRIG^(−/−) mice displayed significantly reducedtumor growth compared to WT mice (p<0.05; FIG. 88A-B). Moreover, PD-L1blockade, begun on day 14, further amplified anti-tumor responses andreduced tumor growth in PVRIG^(−/−) mice compared to anti-PD-L1-treatedWT mice (p=0.052) (FIG. 88C-D). To assess the functional effects ofPD-L1 blockade on PVRIG^(−/−) and WT tumor micro-environments, weharvested tumors and tumor-draining lymph nodes from each of the fourexperimental cohorts on day 18, when groups had received 2 doses ofeither isotype or anti-PD-L1 but no differences in tumor volume wereobserved, and performed flow cytometry for immune subset composition andintracellular cytokines. Immune cell (CD45⁺) trafficking intoPVRIG^(−/−) tumors was enhanced moderately (88% relative to WT tumors)as were CD8⁺ T cells (92% compared to WT tumors) and IFN-γ-producingCD8⁺ T cells (110% increase over WT tumors; FIG. 88E). In combinationwith PD-L1 blockade, infiltration of CD45⁺ cells was increasedsignificantly in PVRIG^(−/−) tumors (160% relative to tumors fromanti-PD-L1-treated WT mice; p=0.032; FIG. 88F). Anti-PD-L1-treatedPVRIG^(−/−) tumors also had greater numbers of total CD8⁺ T cells pertumor weight (252% increase) and IFN-γ-producing CD8+ T cells (297%increase), compared to treated anti-PD-L1 treated WT tumors (FIG. 88F).We also observed that PVRIG^(−/−) mice had unaltered effectortumor-infiltrating CD4⁺ T cell and Foxp3⁺ Treg numbers regardless ofPD-L1 blockade (data not shown). The rescue of immune dysfunction inPVRIG^(−/−) tumors, particularly following PD-L1 blockade, was mirroredin the tumor-draining lymph nodes that had increased frequencies ofIFN-γ⁺TNF-α⁺ effector CD8⁺ T cells relative to anti-PD-L1-treated WTmice (FIG. 88G-H). Taken together, these data demonstrate that PVRIGablation, results in reduced tumor growth associated with an increasedanti-tumor immune response, in particular when combined with anti-PD-L1antibody treatment.

Anti-mPVRIG Antibody Inhibited Tumor Growth in Combination with PDAAntibody or TIGIT Deficiency

After demonstrating that genetic deficiency of PVRIG resulted in reducedtumor growth, we next aimed to demonstrate that antibody-mediatedinhibition of PVRIG-PVRL2 interaction could improve anti-tumor immunity,in particular in combination with PD1 or TIGIT inhibitors as our humanin vitro data has demonstrated. To assess this, we generated a highaffinity, antagonistic anti-mPVRIG antibody. Affinity assessments ofanti-mPVRIG mAb determined by FACS showed sub-nano-molar Kd (0.33 nM onHEK293 mPVRIG, 0.39 nM on D10.G4.1 cells), similar toCHA.7.518.1.H4(S241P) (FIG. 95G-H). The specificity of this antibody wasfurther confirmed as the majority of binding to D10.G4.1 cells wasabrogated upon mPVRIG knockdown (FIG. 95I). Anti-mPVRIG was tested fordisrupting mPVRIG-mPVRL2 interaction by inhibiting the binding of mPVRIGFc to B16F10 and the binding of mPVRL2 Fc to mPVRIG-overexpressingHEK293 cells (FIG. 89A). Complete blocking of PVRIG-PVRL2 interaction byanti-mPVRIG antibody was observed in both assay formats (FIG. 89A, FIG.95J), demonstrating an antagonistic anti-mPVRIG antibody. Next, wetested the in vivo efficacy of mPVRIG blockade in a syngeneic CT26subcutaneous colon tumor model. PVRIG expression was elevated on NK andT cells in the tumor microenvironment, compared to corresponding splenicor draining lymph node subsets (FIG. 89B). Treating tumor bearing micewith anti-mPVRIG blocking mAb as monotherapy failed to reduce tumorgrowth (data not shown). However, combination of anti-PVRIG andanti-PD-L1 mAbs effectively delayed CT26 tumor growth (FIG. 89C) andincreased significantly the survival of treated mice with 40% rate ofcomplete responders (FIG. 89D), Consistent with our human T cell assaydata, these data demonstrate that combination of PD-1 and PVRIGinhibitors can reduce tumor growth.

We also tested the effect of ablating both PVRIG and TIGIT signaling inregulating anti-tumor responses. For these studies, we tested theefficacy of anti-mPVRIG antibody in either WT or TIGIT^(−/−) miceinoculated with B16F10/Db-hmgp100 melanoma cells. Treatment of tumorbearing WT mice with anti-mPVRIG blocking mAb had minor effect comparedto isotype treatment (17% TGI at day 11 and 8% TGI at endpoint, day 18).The effect of TIGIT deletion on tumor growth was minor as well, comparedto WT control group (17% TGI at day 11 and 13% TGI at endpoint).However, when TIGIT deletion was combined with anti-PVRIG mAb treatment,a significant tumor growth inhibition was observed (63% at day 11 and49% TGI at endpoint (FIG. 89E, F). In accordance to tumor growthinhibition, TIGIT^(−/−) mice treated with anti-PVRIG mAb 407 exhibitedincreased survival compared to WT control group, however, statisticalsignificance was not achieved in this aggressive rapidly growing tumormodel (data not shown). Taken together, these data demonstratesynergistic activity of PVRIG inhibitors with PD1 or TIGIT inhibitorsand are in accordance with our human functional data providing therationale for clinical testing of CHA.7.518.1.H4(S241P) with PD1 orTIGIT inhibitors.

Discussion

Although antibodies targeting immune T cell checkpoints such as CTLA4and PD-1 have increased cancer patient survival, the majority of cancerpatients still do not display clinical benefit. One possible reason forthis is the presence of additional T cell regulators that inhibit T cellanti-tumor immunity. Here, we elucidated the role of PVRIG in regulatingeffector T cell function and demonstrate that PVRIG antagonism increasesT cell anti-tumor responses and reduces tumor growth.

PVRIG is a novel member of the nectin and nectin like family, placing itamong several known immunoregulatory receptors in the family.Understanding the interplay of the receptors within this family iscrucial to understanding the relevance and mechanism of action of PVRIG.Of these receptors, DNAM, TIGIT, and CD96 are most closely related toPVRIG in terms of sharing the same ligands, PVR and PVRL2. DNAM binds toboth PVR and PVRL2 and delivers a costimulatory signal to lymphocytes.TIGIT is reported to bind to PVR and weakly to PVRL2. We were unable todetect an interaction between TIGIT and PVRL2 using ELISA or SPR (datanot shown), suggesting that PVR is the dominant ligand for TIGIT. Usingsimilar methods, we and a recent report detected a high affinityinteraction between PVRL2 and PVRIG, suggesting that PVRIG is thedominant inhibitory receptor to PVRL2. These data suggest that TIGIT andPVRIG comprise dual signaling nodes in this axis and that blocking bothis needed for maximal increase of T cell activation within this family.In addition to interacting with different ligands, we observed thatPVRIG has the highest expression on effector or memory T cells, similarto PD-1 whereas TIGIT has the highest expression on regulatory T cells.Furthermore, we observed that PVRIG displayed late induction after Tcell activation as compared to TIGIT. These data suggest that PVRIG hasa unique role within this family, interacting with high affinity toPVRL2 and having a differentiated expression on memory cells and a lateinduction profile to TIGIT.

Reported here is the novel role of PVRIG in regulating anti-tumor T cellresponses using PVRIG deficient mice and antagonistic anti-PVRIGantibodies. It was demonstrated here that mouse PVRIG was expressed on Tcells and NK cells, induced upon lymphocyte activation, and is highestin the TME as compared to the periphery. Furthermore, we show that PVRIGdeficiency led to increased T cell function in-vitro and reduced tumorgrowth in-vivo. An antagonistic antibody to PVRIG reduced tumor growthwhen combined with anti-PD-L1 or genetic deficiency of TIGIT,demonstrating a necessary role of PVRIG in regulating T cell responses.These novel data provide in vivo proof of concept using preclinicaltumor models that targeting PVRIG in combination with PD1 or TIGITantagonism is a potential novel therapy for the treatment of cancers.

Reported here on a high affinity anti-human PVRIG antibody that disruptsthe interaction of PVRIG and PVRL2 which we are pursuing for testing inclinical trials. To determine potential cancer indications that couldinform on patient selection in clinical trials, we examined theexpression profile of this axis in human cancers by FACS and IHC. ForPVRIG, we observed that the mean expression of PVRIG on CD4 and CD8 Tcells by FACS highest in endometrial, lung, kidney, and ovarian cancers,although this difference did not achieve statistical difference withother cancer types as determined by ANOVA with a Tukey's multiplecomparison test with the current number of samples. As PVRIG is inducedupon T cell activation and given that the majority of tumor infiltratingT cells are antigen experienced, it is perhaps not surprising that themedian PVRIG expression was similar across tumor samples and cancertypes. We observed that PVRIG expression was correlated with PD-1 andTIGIT expression, suggesting that the interplay of these 3 inhibitoryreceptors will be important in regulating the anti-tumor response. Inthis report, we observed a synergistic increase in T cell function whenPVRIG antibodies were combined with TIGIT antibodies in a CD8 T celltumor cell co-culture, better than PD-1 in combination with PVRIG orTIGIT inhibitors. These data, along with a previous study demonstratinga role for PVRIG and TIGIT in regulating DC-T cell interactions, showthat this pathway could be involved in regulating T cell-APC and Tcell-tumor cell interactions, and provide multiple mechanisms by whichtargeting PVRIG could increase the anti-tumor immune response.

As expression of PD-L1 has been correlated with clinical response toPD-1 inhibitors, we also analyzed PVRL2 expression in tumors by FACS andIHC to assess whether certain cancer types have higher expression.Assessing dissociated tumor cells, we observed that mean PVRL2expression on macrophages from endometrial, head & neck, kidney, lung,and ovarian samples were higher when compared to other tumor types. MeanPVRL2 expression on CD45⁻ non immune cells was higher on breast,colorectal, endometrial, lung, ovarian, and prostate cancers compared toother cancers. Based on the PVRIG and PVRL2 expression, we determinedthat endometrial, head & neck, lung, kidney, and ovarian cancers have agreater incidence of tumors with high PVRIG and PVRL2 expression andthat these are potential cancers that could response to inhibitors ofthis pathway.

It was observed here that PVRL2 expression can be modulated on antigenproducing cells in vitro by inflammatory mediators whereas PVRL2expression on cancer cells was not altered. These data suggest thatPVRL2 expression on antigen presenting cells can be regulated byinflammation and could be an indicator of an inflamed tumor. Indeed, wedid observe that all PD-L1+ tumors also express PVRL2, both on the tumorcells and in the immune compartment. Expression of PVRL2 on myeloidcells could help predict responses to PVRIG inhibitors in a combinationsetting with PD-1 or TIGIT to further enhance the anti-tumor effect.Interestingly, a portion of PD-L1 negative tumors also expressed PVRL2,primarily on the tumor cells and not on the immune cells. PVR and PVRL2expression on epithelial cells is reported to be induced intumorigenesis, as well as in response to stress and DNA damage. Thesedata are consistent with in vitro findings that the regulation of PVRL2expression on tumor cells is not dependent on IFN-g. As PD-L1 is inducedin an adaptive resistance setting in response to IFN-g and is associatedwith an inflammatory response, the expression of PVRL2 in the absence ofPD-L1 suggests that PVRL2 expression is more prevalent than PD-L1 andthat PVRL2 is expressed in non-inflamed tumors. Based on the above, itis possible that the presence of PVR and PVRL2 contribute to suppressingimmune responses independently of PD-L1 and that inhibitors of PVRIG andTIGIT could be of particular importance in patients that are PD-L1negative or non-responders/progressors to PD-1 inhibitors.

In summary, this report provides several novel insights into PVRIGbiology, including characterizing the expression of this axis in humancancers, demonstrating a prominent role for PVRIG/TIGIT in regulatingthe CD8-tumor cell interaction, and showing that PVRIG antagonism incombination with PD-1 inhibition or TIGIT deficiency lead to asynergistic reduction in tumor growth. These data extend our currentunderstanding of PVRIG biology and provide rationale for clinicaltesting of CHA.7.518.1.H4(S241P), a high affinity anti-PVRIG antibody,in patients with cancer.

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S. Example 19: Tumor Cell Killing Assay

The effect of an anti-human TIGIT antibody and CHA.7.518.1.H4(S241P),either alone or in combination, on tumor cell killing was assessed by anin vitro co-culture assay with human CMV-specific CD8⁺ T cells. TheHLA-A2⁺ target cell lines used in the assay were the melanoma cell line,Mel624, which stably expresses human PVR and PVRL2, and the pancreaticadenocarcinoma cell line, Panc05.04, which expresses endogenous levelsof human PVR and PVRL2. Both tumor cell lines were stably transducedwith a luciferase reporter gene through lentiviral transduction (SystemBiosciences). Mel624 and Panc05.04 cells were pulsed with the CMV pp65peptide at 0.0033 μg/ml or 0.01 μg/ml at 37° C. for 1 hour,respectively. Cells were then washed and plated at 20,000 cells/well. Abenchmark anti-human TIGIT antibody and CHA.7.518.1.H4(S241P) were addedto the culture in combination, or with a control hIgG4 isotype antibodyat 10 μg/ml. Human CMV-specific CD8⁺ T cells from three differentdonors, specified as Donor 4, Donor 72, and Donor 234 were added at100,000 cells/well. Co-cultures were incubated at 37° C. for 16 hours.After the incubation, plates were removed from the incubator and allowedto equilibrate to room temperature for 30 minutes. Bio-Glo luciferasesubstrate (Promega) was added to each well and the mixture equilibratedfor 10 minutes at room temperature protected from light. Luminesce orrelative light units (RLU) was quantified on an EnVision multi-labelreader (Perkin Elmer) with an ultra-sensitive luminescence detector.Percent specific killing was calculated by [(RLU for treatmentantibody—RLU for medium alone)/RLU for medium alone]×100.

Results

FIGS. 99A and B show the effect of the anti-TIGIT antibody andCHA.7.518.1.H4(S241P) treatment on killing of the Mel624 and Panc05.04cells, respectively. When added to the co-culture alone, both theanti-TIGIT antibody and CHA.7.518.1.H4(S241P) induced significant T cellkilling of the tumor cell lines compared to the isotype controlantibody. For the anti-TIGIT antibody the percent specific killingranged from 19-41% for the Mel624 cells, and 3-44% for the Panc05.04cells across the 3 different CMV-reactive donors tested. ForCHA.7.518.1.H4(S241P), the percent specific killing ranged from 16-20%for the Mel624 cells, and 0.21-29% for the Panc05.04 cells. In somecases, an additive effect on tumor cell killing was observed in thecombined treatment of the anti-TIGIT antibody and CHA.7.518.1.H4(S241P).

To determine whether the effect of an anti-TIGIT antibody andCHA.7.518.1.H4(S241P) on tumor cell killing was dose-dependent, theassay was carried out with a 10 point, 2-fold dilution series for eachantibody starting at 0.5 μg/ml for the anti-TIGIT antibodies, and 10μg/ml for CHA.7.518.1.H4(S241P) (FIG. 100). Mel624 killing decreased ina dose-dependent manner when either anti-TIGIT antibody, BM26 orCPA.9.086, were combined with CHA.7.518.1.H4(S241P). More potent killingwas observed for the CPA.9.086 and CHA.7.518.1.H4(S241P) combinationwith an EC₅₀ of 0.40±0.49 nM, compared to the BM26 andCHA.7.518.1.H4(S241P) combination with an EC₅₀ of 2.6±1.7 nM.

T. Example 20: Biophysical Measurement of K_(D)

KinExA equilibrium experiments were performed using a KinExA 3200instrument (Sapidyne Instruments, Boise, Id., USA) at 22° C. RecombinantHis-tagged human TIGIT was obtained from Sino Biologicals (Beijing,China) and reconstituted into 1×PBS. All antigen and antibody samplesfor KinExA analyses were prepared in degassed PBST buffer (PBS with0.05% tween 20) with 100 μg/mL filtered BSA and 0.02% sodium azide. Thesecondary detection antibody used was Alexa Flour 647-labeled goatanti-human IgG (H+L) (Jackson ImmunoResearch Laboratories) diluted 400-to 700-fold in the PBST buffer (with BSA and azide) described above froma 0.5 mg/mL stock in 1×PBS, pH 7.4. For each KinExA experiment, ˜20 μgof human TIGIT was diluted into 1 mL of 50 mM sodium carbonate, pH 9.2which was added directly to 50 mg of azlactone beads (Ultralink Support,Thermo Scientific, Rockford, Ill., USA) and rocked overnight at 4° C.After rocking, the beads were rinsed once with 1 M Tris buffer, pH 8.5,containing 10 mg/mL BSA and rocked for one hour at room temperature inthe same buffer. Coupled beads were added to the bead reservoir in theKinExA instrument and diluted to ˜30 mL with 1×HBS-N (0.01 M Hepes,0.15M NaCl, GE Healthcare) containing 0.02% sodium azide which was alsothe running buffer for the KinExA instrument. All antigen-coupled beadswere used immediately after preparation.

For two replicate measurements of K_(D) for CPA.9.086 (Table 1), 14concentrations of TIGIT ranging from 957 aM-212 pM were equilibrated atroom temperature for ˜72 hours with 2.5 pM CPA.9.086 binding sites and1.8 pM CPA.9.086 binding sites. For CPA.9.083, 14 concentrations ofTIGIT ranging from 478 aM˜196 pM were equilibrated for ˜72 hours with1.8 pM CPA.9.083 binding sites. For duplicate measurements of thebenchmark antibody, BM26 hIgG4, 14 concentrations of TIGIT ranging from9.6 fM-3.53 nM were equilibrated for ˜72 hours with 20 pM BM26 bindingsites and 8.0 pM BM26 binding sites. For CHA.9.547.13, 14 concentrationsof TIGIT ranging from 10.5 fM-2.2 nM were equilibrated for ˜72 hourswith 8 pM mAb CHA.9.547.13 binding sites. The volume flowed through thebead pack for each equilibrated sample for all experiments ranged from 4mL to 11 mL at a flow rate of 0.25 mL/min. Data were fit with a 1:1“standard equilibrium” binding model using KinExA Pro software (Version4.2.10; Sapidyne Instruments) to estimate K_(D) and generate the 95%confidence interval (CI) of the curve fit.

Results

Both CPA.9.083 and CPA.9.086 bound to human TIGIT with femtomolarbinding affinity, while CHA.9.547.13 and BM26 bound with picmolaraffinity. Thus, CPA.9.083 and CPA.9.086 bound to human TIGIT with thehighest affinity of the four different antibodies tested.

TABLE 1 K_(D) measurements of anti-human TIGIT hIgG4 antibodiesdetermined by KinExA Antibody K_(D) ± 95% CI (n = 1) K_(D) ± 95% CI (n =2) CHA.9.547.13 18.8 ± 5.8 pM Not determined CPA.9.083 694 ± 277 fM Notdetermined CPA.9.086 553 ± 230 fM 665 ± 378 fM BM26  8.2 ± 2.8 pM 11.2 ±3.6 pM

U. Example 21: Development and Functional Characterization of CPA.9.086,a Novel Therapeutic Antibody Targeting the Immune Checkpoint TIGIT

Background: TIGIT is a coinhibitory receptor that is highly expressed onlymphocytes, including effector and regulatory CD4+ T cells (Tregs),effector CD8+ T cells, and NK cells, that infiltrate different types oftumors. Engagement of TIGIT with its reported ligands, poliovirusreceptor (PVR) and PVR-like proteins (PVRL2 and PVRL3) directlysuppresses lymphocyte activation. PVR is also broadly expressed intumors, suggesting that the TIGIT-PVR signaling axis may be a dominantimmune escape mechanism for cancer. We report here the biophysical andfunctional characterization of CPA.9.086, a therapeutic antibodytargeting TIGIT. We also demonstrate that co-blockade of TIGIT and a newcheckpoint inhibitor, PVRIG, augments T cell responses.

Materials and Methods: Human phage display and mouse hybridoma antibodydiscovery campaigns were conducted to generate therapeutic anti-TIGITantibodies. The resulting antibodies were evaluated for their ability tobind to recombinant and cell surface-expressed human TIGIT with highaffinity. Cross-reactivity of the antibodies to cynomolgus macaque andmouse TIGIT was also examined. A subset of antibodies that bound withhigh affinity to human TIGIT, and cross-reactive to cynomolgus TIGITwere further characterized for their ability to block the interactionbetween TIGIT and PVR. Blocking antibodies were screened for theirability to enhance antigen-specific T cell activation in vitro eitheralone, or in combination with an anti-PVRIG antibody,CHA.7.518.1.H4(S241P).

Results: A lead antibody, CPA.9.086, was identified that binds to humanTIGIT with high femtomolar affinity. This antibody bound to TIGITendogenously expressed on human CD8+ T cells with higher affinity thantested benchmark antibodies, and was also cross-reactive to bothcynomolgus and mouse TIGIT. When tested for in vitro activity, CPA.9.086augmented cytokine secretion and tumor cell killing by CMV-specific CD8+T cells with superior or equivalent potency to the tested benchmarkantibodies. Combination of CPA.9.086 with an anti-PD1 antibody orCHA.7.518.1.H4(S241P) resulted in enhanced CMV-specific CD8+ T cellactivity. Furthermore, we demonstrated that TIGIT is predominantlyexpressed on Tregs and effector CD8+ T cells from solid tumors comparedto peripheral blood, suggesting that these populations will likely bepreferentially targeted by CPA.9.086.

Conclusion: The development of a very high affinity antagonistic TIGITantibody, CPA.9.086, that is currently in preclinical development isdescribed. We postulate that the femtomolar affinity of CPA.9.086 couldresult in lower and less frequent dosing in patients. CPA.9.086 canenhance human T cell activation either alone or in combination withother checkpoint antibodies. Thus, this data demonstrates the utility oftargeting TIGIT, PD1, and PVRIG for the treatment of cancer.

The invention claimed is:
 1. A composition comprising an antigen bindingdomain that binds to human TIGIT (SEQ ID NO:97) comprising: a) avariable heavy domain comprising SEQ ID NO:160; and b) a variable lightdomain comprising SEQ ID NO:165.
 2. A composition according to claim 1wherein said composition is an antibody comprising: a) a heavy chaincomprising VH-CH1-hinge-CH2-CH3, wherein said VH comprises SEQ IDNO:160; and b) a light chain comprising VL-VC, wherein said VLcomprising SEQ ID NO:165 and VC is either kappa or lambda.
 3. Acomposition according to claim 2 wherein the sequence saidCH1-hinge-CH2-CH3 is selected from human IgG1, IgG2 and IgG4, andvariants thereof.
 4. A composition according to claim 2 wherein saidheavy chain has SEQ ID NO:164 and said light chain has SEQ ID NO:169. 5.A composition according to claim 2 further comprising a second antibodythat binds to a human checkpoint receptor protein.
 6. A compositionaccording to claim 5 wherein said second antibody binds human PD-1.
 7. Acomposition according to claim 5 wherein said second antibody bindshuman PVRIG (SEQ ID NO:2).
 8. A composition according to claim 7 whereinsaid second antibody comprises an antigen binding domain comprising avariable heavy domain comprising SEQ ID NO:5 and a variable light domaincomprising SEQ ID NO:10.
 9. A composition according to claim 7 whereinthe heavy chain of said second antibody has SEQ ID NO:9 and the lightchain of said second antibody has SEQ ID NO:14.
 10. A compositionaccording to claim 7 wherein said second antibody comprises an antigenbinding domain comprising a variable heavy domain comprising SEQ IDNO:15 and a variable light domain comprising SEQ ID NO:20.
 11. Acomposition according to claim 7 wherein the heavy chain of said secondantibody has SEQ ID NO:19 and the light chain of said second antibodyhas SEQ ID NO:24.
 12. A composition according to claim 7 wherein saidsecond antibody comprises: i) the vhCDR1, vhCDR2, and vhCDR3 from SEQ IDNO:5 and ii) the v1CDR1, v1CDR2, and v1CDR3 from SEQ ID NO:10.
 13. Acomposition comprising an antibody that binds to human TIGIT (SEQ IDNO:97) wherein said antibody comprises: i) the vhCDR1, vhCDR2, andvhCDR3 from SEQ ID NO:160; and ii) the v1CDR1, v1CDR2, and v1CDR3 fromSEQ ID NO:165.
 14. A composition according to claim 13 wherein saidantibody comprises a CH1-hinge-CH2-CH3 region from human IgG1, IgG2, orIgG4, or variants thereof.
 15. A composition according to claim 13wherein said antibody comprises a VC region, wherein said VC is eitherkappa or lambda.
 16. A composition according to claim 13 furthercomprising a second antibody that binds to a human checkpoint receptorprotein.
 17. A composition according to claim 16 wherein said secondantibody binds human PD-1.
 18. A composition according to claim 16wherein said second antibody binds human PVRIG (SEQ ID NO:2).
 19. Acomposition according to claim 18 wherein said second antibody comprisesan antigen binding domain comprising a variable heavy domain comprisingSEQ ID NO:5 and a variable light domain comprising SEQ ID NO:10.
 20. Acomposition according to claim 18 wherein said second antibody comprisesan antigen binding domain comprising a variable heavy domain comprisingSEQ ID NO:15 and a variable light domain comprising SEQ ID NO:20.
 21. Acomposition according to claim 18 wherein the heavy chain of said secondantibody has SEQ ID NO:9 and the light chain of said second antibody hasSEQ ID NO:14.
 22. A composition according to claim 18 wherein the heavychain of said second antibody has SEQ ID NO:19 and the light chain ofsaid second antibody has SEQ ID NO:24.